High-affinity oligonucleotide ligands to vascular endothelial growth factor (VEGF)

ABSTRACT

Methods are described for the identification and preparation of nucleic acid ligand ligands to vascular endothelial growth factor (VEGF). Included in the invention are specific RNA ligands to VEGF identified by the SELEX method.

RELATED APPLICATIONS

This application is a Continuation of U.S. patent application Ser. No. 09/156,824, filed Sep. 18, 1998, now abandoned entitled “High Affinity Oligonucleotide Ligands to Vascular Endothelial Growth Factor”, which is a continuation of Ser. No. 08/447169, filed May 19, 1995, now U.S. Pat. No. 5,811,533 entitled “High-Affinity Oligonucleotide Ligands to Vascular Endothelial Growth Factor (VEGF),” which is a Continuation-in-Part of U.S. patent application Ser. No. 07/714,131, filed Jun. 10, 1991, entitled “Nucleic Acid Ligands,” now U.S. Pat. No. 5,475,096, which is a Continuation-in-Part of U.S. patent application Ser. No. 07/536,428, filed Jun. 11, 1990, entitled “Systematic Evolution of Ligands by Exponential Enrichment,” now abandoned. This application is a Continuation-in-Part of U.S. patent application Ser. No. 08/205,515, filed Mar. 3, 1994, now abandoned in favor of U.S. patent application Ser. No. 08/233,012, filed Apr. 25, 1994, entitled “High-Affinity Oligonucleotide Ligands to Vascular Endothelial Growth Factor (VEGF),” and a Continuation-in-Part of U.S. patent application Ser. No. 07/964,624, filed Oct. 21, 1992 entitled “Nucleic Acid Ligands to HIV-RT and HIV-1 Rev,” now U.S. Pat. No. 5,496,938. All applications cited herein are expressly incorporated in their entirety by this reference.

FIELD OF THE INVENTION

Described herein are high affinity nucleic acid ligands to vascular endothelial growth factor (VEGF). The method utilized herein for identifying such nucleic acid ligands is called SELEX, an acronym for Systematic Evolution of Ligands by EXponential enrichment

BACKGROUND OF THE INVENTION

Neovascularization or angiogenesis is the process in which sprouting new blood vessels are formed from the existing endothelium in response to external stimuli that signal inadequate blood supply. Angiogenesis is generally rare under normal physiological conditions but frequently accompanies certain pathological conditions such as psoriasis, rheumatoid arthritis, hemangioma, and solid tumor growth and metastasis (Folkman & Klagsbrun (1987) Science 235:442-447; Kim et al. (1993) Nature 362:841-844). Several growth factors that are capable of inducing angiogenesis in vivo have been identified to date including acidic and basic fibroblast growth factors (aFGF, bFGF), transforming growth factors α and β (TGFα, TGFβ), platelet derived growth factor (PDGF), angiogenin, platelet-derived endothelial cell growth factor (PD-ECGF), interleukin-8 (IL-8), and vascular endothelial growth factor (VEGF).

VEGF was originally purified from guinea pig ascites and tumor cell cultures as a factor that increases vascular permeability (Senger, D. R. et al. (1983) Science 219:983-985) and it has therefore also been referred to as vascular permeability factor (VPF). VEGF is a heat and acid-stable, disulfide-linked homodimer. Four isoforms have been described (121, 165, 189 and 206 amino acids, respectively) and are believed to be the result of alternative splicing of mRNA. Despite the presence of an identical N-terminal hydrophobic signal sequence in all molecular isoforms of VEGF, only the two lower molecular weight species are efficiently secreted (Ferrara, N. et al. (1991) J. Cell. Biochem. 47:211-218). The predominant VEGF isoform in most cells and tissues is the 165 amino acid species. Although VEGF is typically glycosylated, glycosylation is only required for efficient secretion but not for activity (Yeo, T-.K. et al. (1991) Biochem. Biophys. Res. Commun. 179:1568-1575; Peretz, D. et al. (1992) Biochem. Biophys. Res. Commun. 182:1340-1347). The amino acid sequence of VEGF is highly conserved across species and exhibits a modest but significant homology (18-20%) to PDGF A and B (Jakeman L. B. et al. (1992) J. Clin. Invest. 89:244-253; Ferrara et al. (1992) Endocrine Rev. 13:18-32).

Unlike other angiogenic growth factors, target cell specificity of VEGF is limited to vascular endothelial cells. The biological actions of VEGF are mediated through its interaction with specific cell-associated receptors which have been identified in the majority of tissues and organs (Jakeman, L. B. (1992) J. Clin. Invest. 89:244-253). Three high-affinity receptors for VEGF have been cloned to date: flt1, kdr/flk-1 and flt4 (Vaisman, N. et al. (1990) J. Biol. Chem. 265:19461-19466; de Vries, C. et al. (1992) Science 255:989-991; Galland, F. et al. (1993) Oncogene 8:1233-1240). These receptors belong to a family of transmembrane tyrosine kinases and bind VEGF with dissociation constants between 10⁻¹¹ M to 10⁻¹² M. Recent experiments have shown that binding of VEGF to its high-affinity receptors is significantly enhanced by heparin or cell surface-associated heparin-like molecules (Gitay-Goren, H. (1992) J. Biol. Chem. 267:6093-6098).

In addition to promoting the growth of vascular endothelial cells and inducing vascular leakage, VEGF is also known to induce the proteolytic enzymes interstitial collagenase, urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA) (Unemori E. et al. (1993) J. Cell. Physiology 153:557; Pepper, M. S. et al. (1992) Biochem. Biophys. Res. Commun. 189:824). These enzymes are known to play a prominent role in angiogenesis-related extracellular matrix degradation.

As a secreted and specific mitogen for endothelial cells, VEGF may be one of the major angiogenesis inducers in vivo. Several recent observations have supported this notion. For example, the expression of VEGF and its receptors accompanies angiogenesis associated with (i) embryonic development (Breier, G. et al. (1992) Development 114:521-532), (ii) hormonally-regulated reproductive cycle and (iii) tumor growth (Dvorak, H. F. (1991) J. Exp. Med. 174:1275-1278; Shweiki, D. et al. (1992) Nature 359:843-845; Plate, K. H. et al. (1992) Nature 359:845-848). It is relevant to note that aggressive tumor growth is accompanied by the generation of necrotic areas where oxygen and nutrient supplies are inadequate. Oxygen deprivation hypoxia) in tissues is a known angiogenesis stimulant. Interestingly, VEGF expression was found to be the highest in tumor cells facing the necrotic areas (Shweiki, D. et al. (1992) supra; Plate, K. H. et al. (1992) supra). It has therefore been suggested by these authors that VEGF plays a key role in hypoxia-induced angiogenesis.

Recent experiments with neutralizing monoclonal antibodies (MAbs) to VEGF have been especially meaningful for establishing that this growth factor is an important tumor angiogenesis inducer in vivo (Kim, K. J. et al. (1993) Nature 362:841-844). Immunocompromised (nude) mice injected with human rhabdomyosarcoma, glioblastoma or leiomyosarcoma cell lines rapidly develop tumors. Specific neutralizing MAb to VEGF were found to inhibit the growth of these tumors. The density of tumor vasculature was decreased in MAb-treated animals as compared to controls. The same MAb, on the other hand, had no effect on the growth rate of the tumor cells in vitro suggesting that the growth inhibition was not mediated at the cellular level and appears to be mediated by the 165-amino acid isoform of VEGF.

BRIEF SUMMARY OF THE INVENTION

Herein described is the isolation and characterization of binding properties of a set of high-affinity nucleic acid ligands to VEGF. RNA, modified RNA, and ssDNA ligands are provided by the present invention. These ligands were selected from an initial pool of about 10¹⁴ RNA or DNA molecules randomized at thirty or forty contiguous positions. The evolved RNA ligands shown in FIGS. 2A-F bind VEGF with affinities in the low nanomolar range.

Also included herein are modified RNA ligands to VEGF. Such modified RNA ligands may be prepared after the identification of 2′-OH RNA ligands or by performing SELEX using a candidate mixture of modified RNAs. For example, 2′-NH₂ pyrimidine RNA ligands to VEGF are described herein and the evolved ligands are shown in FIG. 9. Additionally post-SELEX modified RNA ligands are provided in Table 4.

Also included herein are ssDNA ligands to VEGF. The evolved ssDNA ligands are shown in Table 8.

The present invention includes the method of identifying nucleic acid ligands and ligand sequences to VEGF comprising:

a) contacting a candidate mixture of nucleic acids with VEGF, wherein nucleic acids having an increased affinity to VEGF relative to the candidate mixture may be partitioned from the remainder of the candidate mixture;

b) partitioning the increased affinity nucleic acids from the remainder of the candidate mixture; and

c) amplifying the increased affinity nucleic acids, whereby nucleic acid ligands to VEGF may be identified.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the starting RNA and PCR primers used in the SELEX experiment described in Examples 1 and 2.

FIGS. 2A-F show the aligned sequences and predicted secondary structures for the six families (grouped by primary sequence homology) of RNA ligands to VEGF. Arrows underline the inverted repeats of the double stranded (stem) regions. Lowercase and uppercase letters are used to distinguish nucleotides in the constant and the evolved sequence regions, respectively. Positions are numbered consecutively starting (arbitrarily) with the evolved nucleotide closest to the 5′ end of the shown window.

FIGS. 3A-F show the consensus sequences and predicted secondary structures for certain of the VEGF ligand families. Plain text is used to designate positions that occur at >60% but <80% frequencies. Positions where individual nucleotides are strongly conserved (frequencies>80%) are outlined. Residues in parenthesis occur at that position with equal frequencies to gaps. The numbering system described in the legend to FIG. 2 is used. R=A or G; Y=C or U; M=A or C; D=A, G or U; V=G, A or C; S=G or C; K=G or U; N=any base and prime (′) indicates a complementary base.

FIGS. 4A-F show the binding curves for a representative set of high-affinity ligands to VEGF. Full-length (∘) and truncated (Δ) ligands tested were 100 (SEQ ID NO:11) and 100t (SEQ ID NO:51) (family 1, FIG. 4A), 44 (SEQ ID NO:20) and 44t (SEQ ID NO:52) (family 2, FIG. 4B), 12 (SEQ ID NO:22) and 12t (SEQ ID NO:53) (family 3, FIG. 4C), 40 (SEQ ID NO:28) and 40t (SEQ ID NO:54) (family 4, FIG. 4D), 84 (SEQ ID NO:36) and 84t (SEQ ID NO:55) (family 5, FIG. 4E), and 126 (SEQ ID NO:38) and 126t (SEQ ID NO:56) (family 6, FIG. 4F). The fraction of ³²P-labeled RNA bound to nitrocellulose filters is plotted as a function of total protein concentration and the lines represent the fit of the data points to eq. 2 (40t, 84 and 84t) or to eq. 5 (all other ligands). RNA concentrations were determined from their absorbance reading at 260 nm (and were typically<50 pM). Binding reactions were done at 37° C. in phosphate buffered saline containing 0.01% human serum albumin

FIGS. 5A and B show the results of the determination of the 3′- and 5′-boundaries for a representative high-affinity VEGF ligand (ligand 12) (SEQ ID NO:50). The 3′-boundary determination (FIG. 5A) showing partially hydrolyzed 5′-end labeled RNA (lane 4), hydrolytic fragments retained on nitrocellulose filters following incubation of the partially hydrolyzed RNA with VEGF at 5 nM (lane 1), 0.5 nM (lane 2), or 0.125 nM (lane 3) and partial digest of the 5′-end labeled RNA with RNAse T₁ (lane 5) resolved on an 8% denaturing polyacrylamide gel. The 5′-boundary (FIG. 5B) was determined in an identical manner except that RNA radiolabeled at the 3′-end was used. Shown are RNase T₁ digest (lane 1), partial alkaline hydrolysis (lane 2), and hydrolytic fragments retained on nitrocellulose filters following incubation with VEGF at 5 nM (lane 3), 0.5 nM (lane 4), or 0.125 nM (lane 5). Arrows indicate the 3′- and the 5′-boundaries of the minimal ligand 12 (italicized).

FIG. 6 shows the Scotchard analysis of ¹²⁵I-VEGF binding to HUVECS. Data points are averages of two determinations. Increasing concentrations of ¹²⁵I-VEGF were incubated with 2×10⁵ cells in the presence or absence of 50-fold excess of unlabeled VEGF to determine the amount of total (∘), specific (□) and non-specific (Δ) binding of ¹²⁵I-VEGF as a function of free ¹²⁵I-VEGF concentration (insert).

FIG. 7 shows the effect of random RNA (∘) and representative high affinity RNA ligands loot (SEQ ID NO:51) (family 1) (Δ) and 44t (SEQ ID NO:52) (family 2) (□) on binding of ¹²⁵-VEGF to cell-surface receptors as a function of RNA concentration. The inhibitory affect of high affinity ligands representing other sequence families is virtually identical to that of ligands 100t and 44t.

FIG. 8 shows the starting random RNAs for experiments A and B, and PCR primers used in identifying 2′-NH,-RNA ligands to VEGF (Example 4).

FIGS. 9A-G show 2′-NH₂-RNA ligands to VEGF identified via the SELEX technology as described in Example 4.

DETAILED DESCRIPTION OF THE INVENTION

This application describes high-affinity nucleic acid ligands to vascular endothelial growth factor (VEGF) identified through the method known as SELEX The SELEX method is described in detail in U.S. patent application Ser. No. 07/536,428, filed Jun. 11, 1990, entitled “Systematic Evolution of Ligands by EXponential Enrichment,” now abandoned, U.S. patent application Ser. No. 07/714,131, filed Jun. 10, 1991, entitled “Nucleic Acid Ligands,” now U.S. Pat. No. 5,475,096, U.S. patent application Ser. No. 07/931,473, filed Aug. 17, 1992, entitled “Methods for Identifying Nucleic Acid Ligands,” now U.S. Pat. No. 5,270,163.

In its most basic form, the SELEX process may be defined by the following series of steps:

1) A candidate mixture of nucleic acids of differing sequence is prepared. The candidate mixture generally includes regions of fixed sequences (i.e., each of the members of the candidate mixture contains the same sequences in the same location) and regions of randomized sequences. The fixed sequence regions are selected either: a) to assist in the amplification steps described below, b) to mimic a sequence known to bind to the target; or c) to enhance the concentration of a given structural arrangement of the nucleic acids in the candidate mixture. The randomized sequences can be totally randomized (i.e., the probability of finding a base at any position being one in four) or only partially randomized (e.g., the probability of finding a base at any location can be selected at any level between 0 and 100 percent).

2) The candidate mixture is contacted with the selected target under conditions favorable for binding between the target and members of the candidate mixture. Under these circumstances, the interaction between the target and the nucleic acids of the candidate mixture can be considered as forming nucleic acid-target pairs between the target and those nucleic acids having the strongest affinity for the target.

3) The nucleic acids with the highest affinity for the target are partitioned from those nucleic acids with lesser affinity to the target. Because only an extremely small number of sequences (and possibly only one molecule of nucleic acid) corresponding to the highest affinity nucleic acids exist in the candidate mixture, it is generally desirable to set the partitioning criteria so that a significant amount of the nucleic acids in the candidate mixture (approximately 5-50%) are retained during partitioning.

4) Those nucleic acids selected during partitioning as having the relatively higher affinity to the target are then amplified to create a new candidate mixture that is enriched in nucleic acids having a relatively higher affinity for the target

5) By repeating the partitioning and amplifying steps above, the newly formed candidate mixture contains fewer and fewer unique sequences, and the average degree of affinity of the nucleic acids to the target will generally increase. Taken to its extreme, the SELEX process will yield a candidate mixture containing one or a small number of unique nucleic acids representing those nucleic acids from the original candidate mixture having the highest affinity to the target molecule.

The SELEX Patent Applications describe and elaborate on this process in great detail. Included are targets that can be used in the process; methods for the preparation of the initial candidate mixture; methods for partitioning nucleic acids within a candidate mixture; and methods for amplifying partitioned nucleic acids to generate enriched candidate mixtures. The SELEX Patent Applications also describe ligand solutions obtained to a number of target species, including both protein targets where the protein is and is not a nucleic acid binding protein.

SELEX provides high affinity ligands of a target molecule. This represents a singular achievement that is unprecedented in the field of nucleic acids research. The present invention applies the SELEX procedure to the specific target of vascular endothelial growth factor (VEGF). In the Example section below, the experimental parameters used to isolate and identify the nucleic acid ligand solutions to VEGF are described.

In order to produce nucleic acids desirable for use as a pharmaceutical, it is preferred that the nucleic acid ligand 1) binds to the target in a manner capable of achieving the desired effect on the target; 2) be as small as possible to obtain the desired effect; 3) be as stable as possible; and 4) be a specific ligand to the chosen target. In most situations it is preferred that the nucleic acid ligand have the highest possible affinity to the target.

In co-pending and commonly assigned U.S. patent application Ser. No. 07/964,624, filed Oct. 21, 1992, now U.S. Pat. No. 5,496,938 ('938 patent), methods are described for obtaining improved nucleic acid ligands after SELEX has been performed. The '938 patent, entitled “Nucleic Acid Ligands to HIV-RT and HIV-1 Rev” is specifically incorporated herein by reference.

This invention includes the specific RNA ligands to VEGF shown in FIGS. 2A-F (SEQ ID NOS:4-38). The scope of the ligands covered by this invention extends to all RNA ligands of VEGF identified according to the SELEX procedure. More specifically, this invention includes nucleic acid sequences that are substantially homologous to and that have substantially the same ability to bind VEGF as the specific nucleic acid ligands shown in FIGS. 2A-F. By substantially homologous it is meant a degree of primary sequence homology in excess of 70%, most preferably in excess of 80%. Substantially the same ability to bind VEGF means that the affinity is within one order of magnitude of the affinity of the ligands described herein. It is well within the skill of those of ordinary skill in the art to determine whether a given sequence—substantially homologous to those specifically described herein—has substantially the same ability to bind VEGF.

This invention also includes the 2′-NH₂ modified RNA ligands to VEGF as shown in FIGS. 9A-G (SEQ ID NOS:63-146). The scope of the present invention extends, therefore, to all modified nucleic acid ligands identified according to the SELEX method as well as to all sequences that are substantially homologous to and that have substantially the same ability to bind VEGF as ligands predicted in FIGS. 9A-G.

This invention also includes additional post-SELEX modified RNA ligands having 2′-O-methyl groups on various purine residues. In addition, nucleotides that contain phosphorothioate backbone linkages were added at the 5′ and 3′ ends of the ligands in order to reduce or prevent degradation by exonucleases. Internal backbone positions were also identified in which phosphorothioate linkages could be substituted, without the loss of binding affinity, to reduce or prevent endonucleolytic degradation. The post-SELEX modified RNA ligands provided in Table 4 (SEQ ID NOS:147-158) demonstrate an ability to inhibit the activity of exonucleases and endonucleases, without affecting binding affinities.

Further, this invention includes ssDNA ligands to VEGF. The specific ssDNA ligands are shown in Table 8 (SEQ ID NOS:159-230).

The scope of the ligands covered by this invention extends to all ssDNA ligands of VEGF identified according to the SELEX procedure. More specifically, this invention includes nucleic acid sequences that are substantially homologous to and that have substantially the same ability to bind VEGF as the specific nucleic acid ligands shown in Table 8. By substantially homologous it is meant a degree of primary sequence homology in excess of 70%, most preferably in excess of 80%. Substantially the same ability to bind VEGF means that the affinity is within one order of magnitude of the affinity of the ligands described herein. It is well within the skill of those of ordinary skill in the art to determine whether a given sequence—substantially homologous to those specifically described herein—has substantially the same ability to bind VEGF.

This invention encompasses the use of the disclosed ligands to identify a second ligand. In one embodiment, a first SELEX identified ligand which binds to a specific site of the target molecule is used to elute secondary ligands binding to the same site. In another embodiment, a first SELEX identified ligand binding to a specific site of the target molecule is used to select secondary ligands which do not bind to the same site. In this case, SELEX is conducted in the presence of the first ligand such that the binding site is saturated with the first ligand and selection occurs for ligands binding elsewhere on the target molecule. In a further embodiment analogous to the generation of anti-idiotype antibodies, a SELEX identified ligand to VEGF may itself be used as a target molecule to identify secondary ligands resembling the VEGF binding site. Such secondary ligands may compete with VEGF-substrate binding and inhibit the biological activity of VEGF.

A review of the sequence homologies of the nucleic acid ligands of VEGF shown in FIGS. 2A-F and 9A-G and Table 8 shows that sequences with little or no primary homology may have substantially the same ability to bind VEGF. For this reasons, this invention also includes nucleic acid ligands that have substantially the same structure as the ligands presented herein and the substantially the same ability to bind VEGF as the nucleic acid ligands shown in FIGS. 2A-F and 9A-G and Table 8.

The following examples are provided to explain and illustrate the present invention and are not to be taken as limiting of the invention.

Example 1 describes the experimental procedures used to generate high-affinity nucleic acid ligands to VEGF. Example 2 describes the high-affinity RNA ligands to VEGF shown in FIGS. 2A-F. Example 3 describes the specificity of truncated RNA ligands to VEGF. Example 4 describes the experimental procedures used to generate 2′-NH₂ pyrimidine modified RNA ligands to VEGF. Example 5 describes post-SELEX modifications of VEGF RNA ligands with 2′-O-methyl groups on purines. Additionally, phosphorothioate backbone substitutions were made to reduce or prevent nuclease degradation without effecting binding affinity. Example 6 describes the stability of post-SELEX modified VEGF RNA ligands to ex vivo rat tissue degradation. Example 7 describes obtaining ssDNA ligands to VEGF.

EXAMPLE 1 Experimental Procedures

Materials. Recombinant human VEGF (165 amino acid form; MW 46,000) was a generous gift from Dr. Napoleone Ferrara (Genentech). All other reagents and chemicals were of the highest purity available and were purchased from commercial sources.

SELEX Essential features of the SELEX protocol have been described in detail in U.S. Pat. No. 5,270,163 as well as in previous papers from these laboratories (See, e.g., Schneider et al. (1992) J. Mol. Biol. 228:862). Briefly, DNA templates for in vitro transcription (that contain a region of thirty random positions flanked by constant sequence regions) and the corresponding PCR primers were prepared chemically using established solid phase oligonucleotide synthesis protocols.

The random region was generated by utilizing an equimolar mixture of the four unmodified nucleotides during oligonucleotide synthesis. The two constant regions were designed to contain PCR primer annealing sites, primer annealing site for cDNA synthesis, T7 RNA polymerase promoter region and restriction enzyme sites that allow cloning into vectors (FIG. 1) (SEQ ID NOS:1-3). An initial pool of RNA molecules was prepared by in vitro ascription of approximately 200 picomoles (10¹⁴ molecules) of the double stranded DNA template utilizing T7 RNA polymerase. Transcription mixtures consisting of 100-300 nM template, 5 units/μl T7 RNA polymerase, 40 mM Tris-Cl buffer (pH 8.0) containing 12 mM MgCl₂, 5 mM DTT, 1 mM spermidine, 0.002% Triton X-100, 4% PEG were incubated at 37° C. for 2-3 hours. These conditions typically resulted in transcriptional amplification of 10 to 100-fold. Selections for high affinity RNA ligands were done by incubating VEGF with RNA for 10-20 minutes at 37° C. in 50 ml of phosphate buffered saline (PBS=10.1 mM Na₁HPO₄, 1.8 mM KH₂PO₄, 137 mM NaCl, 2.7 mM KCl, pH 7.4) and then separating the protein-RNA complexes from the unbound species by nitrocellulose filter partitioning (Tuerk, C. and Gold, L. (1990) Science 249:505-510). The selected RNA (which typically amounted to 5-10% of the total input RNA) was then extracted from the filters and reverse transcribed into cDNA by avian myeloblastoma virus reverse transcriptase (AMV RT). Reverse transcriptions were done at 48° C. (60 min) in 50 mM Tris buffer (pH 8.3), 60 mM NaCl, 6 mM Mg(OAc)₂, 10 mM DTT and 1 unit/μl AMV RT. Amplification of the cDNA by PCR under standard conditions yielded a sufficient amount of double-stranded DNA for the next round of in vitro transcription.

Nitrocellulose Filter Binding Assays. Oligonucleotides bound to proteins can be effectively separated from the unbound species by filtration through nitrocellulose membrane filters (Yarus, M. and Berg, P. (1970) Anal. Biochem. 35:450-465; Lowary, P. T. and Uhlenbeck, O. C. (1987) Nucleic Acids Res. 15:10483-10493; Tuerk, C. and Gold, L. (1990) supra). Nitrocellulose filters (0.2 μm pore size, Schleicher and Schuell, Keene, N H) were secured on a filter manifold and washed with 4-10 ml of buffer. Following incubations of ³²P labeled RNA with serial dilutions of the protein for 10 min at 37° C. in buffer (PBS) containing 0.01% human serum albumin (HSA), the solutions were applied to the filters under gentle vacuum in 45 ml aliquots and washed with 5 ml of PBS. The filters were then dried under an infrared lamp and counted in a scintillation counter.

Equilibrium Dissociation Constants. In the simplest case, equilibrium binding of RNA (R) to VEGF (P) can be described by eq. 1, $\begin{matrix} {{R \cdot P}\overset{Kd}{\rightleftharpoons}{R + P}} & (1) \end{matrix}$

where Kd=([R][P]/[R.P]) is the equilibrium dissociation constant Using the mass-balance equations, the fraction of bound RNA at equilibrium (q) can be expressed in terms of measurable quantities (eq. 2),

q=(f2Rt){Pt+Rt+Kd−[(Pt+Rt+Kd)²−4PtRt] ^(½)}  (2)

where Pt and Rt are total protein and total RNA concentrations and f reflects the efficiency of retention of the protein-RNA complexes on nitrocellulose filters. The average value of f for VEGF in our assays was 0.7.

Most RNA ligands exhibited biphasic binding to VEGF. For those ligands, binding of RNA to VEGF is described by a model where total RNA is assumed to be partitioned between two non-interconverting components (R1 and R2) that bind to VEGF with different affinities (eqs 3 and 4). $\begin{matrix} {{{R1} \cdot P}\overset{Kd1}{\rightleftharpoons}{{R1} + P}} & (3) \\ {{{R2} \cdot P}\overset{Kd2}{\rightleftharpoons}{{R2} + P}} & (4) \end{matrix}$

In this case, the fraction of total bound RNA (q) is given by eq. 5.

q=(f/2Rt){2Pt+Rt+Kd1+Kd2−[(Pt+χ1Rt+Kd1)²−4Ptχ1Rt] ^(½)−[(Pt+χ2Rt+Kd2)²−4Ptχ2Rt] ^(½)}  (5)

where χ1 and χ2(=1-c1) are the mole fractions of R1 and R2 and Kd1 and Kd2 are the corresponding dissociation constants.

Internally-labeled RNA ligands used for binding studies were prepared by in vitro transcription using T7 RNA polymerase (Milligan et al. (1987) Nucl. Acids Res. 15:8783) and were purified on denaturing polyacrylamide gels to ensure size homogeneity. All RNA ligands were diluted to about 1 nM in PBS, denatured at 90° C. for 2 minutes, and then cooled on ice prior to incubation with the protein. This denaturation/renaturation cycle performed at high dilution is necessary to ensure that the RNA is essentially free from dimers and other higher order aggregates. Concentrations of the stock solutions of VEGF, from which other dilutions were made, were determined from the absorbance reading at 280 nm using the calculated value for ε₂₈₀ of 46,600 M⁻¹ cm⁻¹ for the VEGF dimer (Gill et al. (1989) Anal. Biochem. 182:319). Data sets that define the binding curves were fit to either eq. 2 or eq. 5 by the non-linear least squares method using the software package Kaleidagraph (Synergy Software, Reading, Pa.).

Information Boundary Determinations. High-affinity VEGF ligands were radiolabeled at the 5′-end with γ-³²P-ATP (New England Biolabs, Beverly, Mass.) and T4 polynucleotide kinase (New England Biolabs, Beverly, Mass.) for the 3′-boundary determinations, or at the 3′-end with α-³²PCp and T4 RNA ligase (New England Biolabs) for the 5′-boundary determination. Radiolabeled RNA ligands were subjected to partial alkaline hydrolysis and then selectively bound in solution to VEGF at 5, 0.5, or 0.125 nM before being passed through nitrocellulose filters. Retained oligonucleotides were resolved on 8% denaturing polyacrylamide gels. In each experiment, the smallest radiolabeled oligonucleotide bound by VEFG at the lowest protein concentration defines the information boundary. Partial digests of the 5′- or the 3′-labelled RNA ligands with RNAse T₁ (Boehringer Mannheim Biochemicals, Indianapolis, Ind.) were used to mark the positions of labeled oligonucleotides ending with a guanosine.

Cloning and Sequencing. Individual members of the enriched pool were cloned into pUC18 vector and sequenced as described (Schneider, D. et al. (1992) J. Mol. Biol. 228:862-869).

Receptor Binding. VEGF was radioiodinated by the Iodegen method (Jakeman et al. (1992) J. Clin. Invest. 89:244) to a specific activity of 2.4×10⁴ cpm/ng. Human umbilical vein endothelial cells (HUVECs) were plated in 24-well plates at a density of 1-2×10⁵ cells/well and grown to confluence in EGM (Clonetics, San Diego, Calif.) media (24-48 hours). At confluence, the cells were washed 3 times with PBS and incubated for 2 hrs at 4° C. in α-MEM serum-free media containing ¹²⁵I-labeled VEGF with or without unlabeled competitor (VEGF, EGF, or RNA). For experiments done with RNA, 0.2 units of placental RNase inhibitor (Promega, Madison, Wis.) were included in the media It was determined that the RNA ligands were not degraded during the course of the experiment. At the end of the 2 hour incubation period, the supernatant was removed and the wells washed 2 times with PBS. HUVECs were then lysed with 1% triton X-100/1 M NaOH and the amount of cell-associated ¹²⁵I-VEGF determined by gamma counting.

EXAMPLE 2 RNA Ligands to VEGF

Approximately 10¹⁴ RNA molecules randomized at third contiguous positions (FIG. 1) (SEQ ID NO:1) were used in the initial selection targeting VEGF. Random RNA bound to VEGF with an affinity of approximately 0.2 μM. After 13 rounds of SELEX, the observed improvement in affinity of the evolved RNA pool was about two orders of magnitude (data not shown). 64 isolates were cloned and sequenced from this evolved pool, and 37 unique sequences found (sequences differing at only one or two positions were not considered unique). 34 of the 37 unique sequences could be classified into six families based on sequence similarity in the evolved region (FIGS. 2A-F) (SEQ ID NOS:4-38). The evolved sequence is provided in capitol letters in FIG. 2. Lower case letters indicate portions of the fixed sequence included in the alignment. The cloned sequence included both the evolved and fixed sequences. Three unique clones, 4 (GGGAUGUUUGGCUAUCUCGGAUAGUGCCCC)(SEQ ID NO:39), 16 (GCUUAAUACGACUCACUNUAGGGAGCUCAG)(SEQ ID NO:40) and 18 (UUGAGUGAUGUGCUUGACGUAUCGCUGCAC)(SEQ ID NO:41) had a more limited sequence similarity with members of the six families.

Consensus Structures. In addition to allowing determination of consensus primary structures, groups of similar sequences consisting of members that share a defined functional property often contain useful clues for secondary structure prediction (James et al. (1989) Meth. Enzymol. 180:227). The underlying assumption is that ligands with similar primary structures are capable of adopting similar secondary structures in which the conserved residues are organized in unique, well-defined motifs. In this context, ligands which have strong, unambiguous secondary structures can provide good structural leads for other sequences within a similar set where consensus folding may be less obvious. Conserved elements of secondary structure, such as base-pairing, may also be detected through covariation analysis of aligned sequence sets (James et al. (1989) supra; Gutell et al. (1992) Nucl. Acids Res. 20:5785). The predicted consensus secondary structures for the six sequence families are shown in FIGS. 3A-F (SEQ ID NOS:42-47).

The most highly conserved residues in the family 1 sequence set (A17, G19 and the CAUC sequence at positions 23-26) can be accommodated in the 9-10 nucleotide loop (SEQ ID NO:42). Base-pairing covariation between positions 16 and 27 (G-C occurs with a frequency of 8 out of 11 times (8/11) and C-G with a frequency of 3/11), positions 15 and 28 (U-G, 7/1 1; G-C, 3/1 1; U-A, 1/11) and positions 14 and 29 (G-C, 511 1; U-A, 21 1, and C-G, 1/11) supports the predicted secondary structure. It is worth noting that many ligands in this family have stable extended stems that contain up to 15 base pairs.

In the family 2 sequence set, the strongly conserved UGCCG and UUGAUG(G/U)G sequences (positions 8-12 and 26-33) are circularly permutated. In the consensus secondary structures, these nucleotides are found in an identical arrangement within or adjacent to the asymmetrical internal loop (FIG. 3A) (SEQ ID NO:43). This result suggests that the nucleotides outside of the consensus motif shown in FIGS. 3A-F are unimportant for binding. Base-pairing covariation is noted between positions 5 and 36 (C-G, 2/7; G-C, 2/7; U-A, 1/7; G-U, 1/7), 6 and 35 (A-U, 4/7; C-G, 1/7; G-C, 1/7), 7 and 34 (A-U, 4/7; G-C, 1/7), 11 and 28 (C-G, 6/7; G-C, 1/7), 12 and 27 (G-U, 6/7; C-G, 1/7), 13 and 26 (A-U, 5/7; G-C, 1/7; G-U, 1/7), 14 and 25 (G-C, 4/7; C-G, 2/7) and 15 and 24 (C-G, 4/7; G-C, 217).

Family 3 and family 4 sequence sets are characterized by highly conserved contiguous stretches of 21 (GGGAACCUGCGU(C/U)UCGGCACC (SEQ ID NO:48), positions 11-31) and 15 (GGUUGAGUCUGUCCC (SEQ ID NO:49), positions 15-29) arranged in bulged hairpin motifs (FIGS. 3C and D) (SEQ ID NOS:44-45). Base-pairing covariation is detected in family 3 between positions 8 and 33 (A-U, 2/4; G-C, 2/4), 9 and 32 (A-U, 2/4; U-A, 1/4; G-C, 1/4), and 10 and 31 (A-U, 1/4; G-C, 3/4) and in family 4 between positions 13 and 31 (A-U, 4/7; C-G, 2/7; U-A, 1/7) and 14 and 30 (C-G, 3/7; U-A, 3/7; A-U, 1/7).

Family 5 consensus secondary structure is an asymmetrical internal loop where the conserved UAGUUGG (positions 9-15) and CCG (positions 29-31) sequences are interrupted by less conserved sequences (FIG. 3E) (SEQ ID NO:46). Modest base-pairing covariation is found between positions 8 and 32 (A-U, 2/4; U-G, 1/4), 16 and 26 (G-C, 2/4; A-U, 1/4), 17 and 25 (A-U, 2/4; G-C, 1/4) and 18 and 24 (C-G, 2/4; G-C, 1/4).

Family 6 has only two sequences and therefore the concept of consensus sequence or consensus structure is less meaningful. Nevertheless, the two sequences are very similar (90% identity) and can be folded into a common motif (FIG. 3F) (SEQ ID NO:47). Base-pairing covariation is found between positions 1 and 32 (A-U, 112; G-U, 1/2), 2 and 31 (C-G, 1/2; G-C, 1/2), 14 and 20 (U-A, 1J2; G-C, 1/2) and 15 and 19 (A-U, 1/2; G-U, 1/2).

Affinities. The affinity of all unique sequence clones for VEGF was screened by determining the amount of RNA bound to VEGF at two protein concentrations (1 and 10 nM). Binding of the best ligands from each of the six sequence families was then analyzed over a range of protein concentrations (FIGS. 4A-F). Dissociation constants were calculated by fitting the data points to either eq. 2 (monophasic binding) or eq. 5 (biphasic binding) and their values are shown in Table 1.

Information Boundaries. In order to determine the minimal sequence information necessary for high-affinity binding to VEGF, deletion analyses were performed with representative members from each of the six families. These experiments were done by radiolabeling RNA ligands at either the 3′ end or the 5′ end (for the 3′ or the 5′ boundary determinations, respectively) followed by limited alkaline hydrolysis, partitioning of the free and the bound RNA by nitrocellulose filtration and resolving the hydrolytic fragments that retained high affinity for VEGF on denaturing polyacrylamide gels (Tuerk et al. (1990) J. Mol. Biol. 213:749). The combined information from the 3′ and the 5′ boundary experiments outlines the shortest sequence segment that has high affinity for the protein (FIG. 5) (SEQ ID NO:50). It is important to realize that these experiments define boundaries sequentially at the unlabeled ends of ligands in the context of full-length labeled ends. Since the full-length ends may provide additional contacts with the protein or participate in competing secondary structures, ligands truncated at both ends may have lower or higher affinities for the protein than their full-length parent. The following truncated ligands were prepared by in vitro transcription from synthetic DNA templates: 100t (Family 1) GGCCGGUAGUCGCAUGGCCCAUCGCGCCCGG (SEQ ID NO:51), 44t (Family 2) GGaaGCUUGAUGGGUGACACACGUCAUGCCGAGCu (SEQ ID NO:52), 12t (Family 3) GGAAGGGAACCUGCGUCUCGGCACCuucg (SEQ ID NO:53), 40t (Family 4) GGUCAACGGUUGAGUCUGUCCCGuucgac (SEQ ID NO:54), 84t (Family 5) GgcucaaUAGUUGGAGGCCUGUCCUCGCCGUAGAGC (SEQ ID NO:55) and 126t (Family 6) GGaACGGUUCUGUGUGUGGACUAGCCGCGGCCGuu (SEQ ID NO:56) (letter t designates truncated sequences; underlined guanines are not present in the original sequences and were added to increase the transcriptional efficiency (Milligan et al. (1990) supra); lowercase letters indicate nucleotides from the constant sequence region). Binding curves for these truncated ligands and their dissociation constants are shown alongside their parent ligands in FIGS. 4A-F and Table 1. The dissociation constants of the truncated versus full-length ligands are generally comparable, although ligands 40t (SEQ ID NO:54) and 126t (SEQ ID NO:56) clearly bind to VEGF significantly less well than the corresponding full-length ligands.

Competition experiments revealed that binding of all possible pairwise combinations of truncated ligands representing each of the families is mutually exclusive (lot, 44t, 12t, 40t, 84t and 126t (SEQ ID NOS:51-56, respectively). Furthermore, all of these ligands are displaced by low-molecular weight (≅5,100 Da) heparin (data not shown). Truncated ligands and low-molecular weight heparin were used in these studies in order to maximize the probability of observing non-competing ligand pairs. It appears, therefore, that although there are multiple non-isomorphic solutions to high-affinity binding to VEGF, all examined ligands may bind to the same region of the protein. Proteins in general may have “immunodominant” domains for nucleic acid ligands.

EXAMPLE 3 Specificity of Truncated RNA Ligands to VEGF

Binding of two truncated high-affinity ligands, 100t and 44t (SEQ ID NOS:51-52), to four other heparin binding proteins (bFGF, PDGF, antithrombin III and thrombin) was tested in order to address the question of specificity. Dissociation constants were determined using the nitrocellulose filter partitioning technique. Results are shown in Table 2. Binding of these ligands to VEGF in a buffer containing 10 mM dithiothreitol is at least 1000-fold weaker.

Receptor Binding. Unlabeled VEGF but not EGF was shown to inhibit binding of ¹²⁵I-VEGF to HUVECs in a concentration-dependent manner (data not shown), confirming that ¹²⁵I-VEGF binds to specific sites on HUVECs. As previous studies have reported (Myoken et al. (1991) Proc. Natl. Acad. Sci. USA 88:5819), two classes of receptors on HUVECs were observed to bind VEGF with dissociation constants of ˜5×10⁻¹¹ M (7,000 receptors/cell) and ˜5×10⁻M (20,000 receptors/cell) (FIG. 6).

A group of truncated RNA ligands representing each of the sequence families (lOOt, family 1; 44t, family 2; 12t, family 3; 40t, family 4; 84t, family 5; and 126t, family 6 (SEQ ID NOS:51-56)), as well as random RNA were tested for their ability to inhibit binding of VEGF to its cell-surface receptors. All high-affinity ligands, but not random RNA, inhibited VEGF-VEGF receptor interaction in a concentration-dependent manner with half-inhibition occurring in the 20-40 nM range (FIG. 7).

EXAMPLE 4 Modified 2′-NH₂ Pyrimidine RNA Ligands to VEGF

In order to generate ligands with improved stability in vivo, two SELEX experiments (A and B) targeting VEGF were initiated with separate pools of randomized RNA containing amino (NH₂) functionalities at the 2′-position of each pyrimidine. Starting ligand pools for the two experiments contained approximately 10¹⁴ molecules (500 pmols) of modified RNA randomized at 30 (SELEX experiment A) and 50 (SELEX experiment B) contiguous positions. The starting RNAs and the corresponding PCR primers are defined in FIG. 8 (SEQ ID NOS:57-62). Sequences corresponding to the evolved regions of modified RNA are shown in FIGS. 9A-G.

Ligands with similar primary structures were grouped into 5 families and their consensus sequences are shown below each sequence set FIGS. 9A-G (SEQ ID NOS:63-146). Groups of sequences with similar primary structure (families) have been aligned in FIGS. 9A-G and their consensus sequences are shown below each set Pairs of similar/related sequences, sequences that could not be included in any of the families (“other sequences”) and sequences that correspond to ligands that bind additionally to nitrocellulose filters with high affinity have been shown in separate groups. Letter N in a sequence indicates an ambiguous position on a sequencing gel. Italicized letter N in a consensus sequence indicates a position that is not conserved (i.e., any nucleotide may be found at that position). Dissociation constants for Random RNA A (30N8), Random RNA B (50N7) and a set of modified (2′-amino pyrimidine high-affinity RNA ligands for VEGF are shown in Table 3.

EXAMPLE 5 Post SELEX Modifications of VEGF RNA Ligands

In an attempt to further stabilize the nucleic acid ligands of the invention, certain post-SELEX modifications were done. The ligand NX107 (SEQ ID NO:147) was chosen as a model for post-SELEX modification. NX107 is a truncated version of Ligand 24A (SEQ ID NO:79) from Example 4. All of the pyrimidines in NX107 have an NH₂ group substituted at the 2′-position of the ribose. This example describes substitution of O-Methyl groups at the 2′-position of the ribose of certain of the purines of NX107. Additionally, phosphorothioate nucleotides were added at the 5′ and 3′ ends of the ligands and in at least one instance, at an internal position. The various substitutions to the ligand were designed to inhibit the activity of exonucleases and endonucleases, but not affect binding affinity.

To this end, certain ligands were synthesized and tested for binding affinity. The sequences and the results of the binding studies are provided in Table 4. The binding studies were performed using the protocols described in Example 1.

EXAMPLE 6 Stability of Post-SELEX Modified VEGF Ligands to Ex Vivo Rat Tissue Degradation

In order to be able to quickly assess the effects of ligand modifications on stability to tissue nucleases, the following assay was developed. Brain, kidney, liver and spleen tissues were removed from a freshly sacrificed rat, washed in saline to remove blood, and sliced into approximately 10 mm³ pieces. Each piece was put into an Eppendorf tube with 50 μl PBS and quick frozen on dry ice. Tissues from the same rat were used for all the experiments described here. The ligand to be tested was 5′end-labeled with ³²P, added to the thawed tissue slice in 80 μl PBS, and incubated at 37° C. Aliquots were withdrawn at 3, 10, 30, and 60 minutes, added to an equal volume of formamide dyes on ice, and quick-frozen on dry ice. The samples were run on a 20% denaturing acrylamide gel along with equal counts of the unincubated ligand, and a partial alkaline hydrolysate of the ligand (or a related ligand) for sequence markers. The gels were dried and exposed to X-ray film and a phosphorimager plate (for quantitation of degradation).

The VEGF ligands used in this study are shown in Table 4. Each ligand has the same core 24-mer sequence derived from a truncated 2′NH₂-pyrimidine SELEXed ligand (NX-107)(SEQ ID NO:147). NX-178 (SEQ ID NO:149) is the same 2′amino pyrimidine ligand with phosphorothioate backbone linked thymidine caps at the 5′- and 3′-ends of the ligand. NX-190 (SEQ ID NO:150) is an all DNA version of the same sequence with the above-described caps, and NX-191 (SEQ ID NO:151) is an all 2′OMe version. NX-213 (SEQ ID NO:152) is the capped amino ligand with all the purines 2′OMe substituted except four. NX-2 15 (SEQ ID NO:154) is the same as NX-213 with an internal phosphorothioate linkage between A7 and U8.

Tables 5 and 6 provide the results obtained by this assay on rat brain and kidney tissues as indicated by the percent of full length material found at the various time points. For this analysis, a ligand is still considered functionally intact with cuts in the phosphorothioate caps. The other tissues assayed had similar results. The post-SELEX modifications were successful in protecting the ligand from various endo- and exonucleases.

EXAMPLE 7 SSDNA Ligands to VEGF

This example demonstrates the ability to obtain ssDNA ligands to vascular endothelial growth factor (VEGF).

Most of the materials and methods are the same as those described in Example 1. Two libraries of synthetic DNA oligonucleotides containing 40 random nucleotides flanked by invariant primer annealing sites were amplified by the Polymerase Chain Reaction (CR) using oligonucleotide primers as shown in Table 7 (SEQ ID NOS:237-242). The protocols for the SELEX procedure are as described by Jellinek et al. (PNAS (1993) 90:11227-11231), in the SELEX Patent Applications and in Example 1. VEGF protein binding assays, receptor binding assays, and information boundary determinations are also described in Example 1.

The ssDNA ligands identified are shown in Table 8 (SEQ ID NOS:159-220). Only the sequence of the evolved region is provided in Table 8, however, each of the clones also includes the fixed regions of either SEQ ID NO:237 or SEQ ID NO:240. Clones named with numbers only include the fixed regions of SEQ ID NO:237 and clones named with b and number included the fixed regions of SEQ ID NO:240. Truncations (information boundary determinations) were performed on a number of ligands, which is also provided in Table 8 (SEQ ID NOS:221-230). Four sequence families were obtained from the alignment of the primary sequences of these ligands and a consensus sequence generated for each family (SEQ ID NOS:231-236). Orphan sequences were also identified. Select ligands were tested in the VEGF protein binding assay with results being shown in Table 8. The starting DNA random pool had a binding affinity Kd of approximately 200 nM. In the VEGF receptor binding assay, the truncated clone 33t (SEQ ID NO:224) had a Ki of 3 nM.

TABLE 1 Dissociation Constants For a Representative Set of Full-Length and Truncated High-Affinity RNA Ligands for VEGF.^(a) SEQ ID LIGAND^(b) Kd1 (nM)^(c) χ¹ ^(d) Kd2 (nM)^(e) NOS. 100 0.20 ± 0.02 0.82 ± 0.02 42 ± 30 11 100t 0.42 ± 0.04 0.76 ± 0.03 182 ± 94  51  44 1.7 ± 0.5 0.70 ± 0.11 38 ± 32 20  44t 0.48 ± 0.04 0.73 ± 0.01 82 ± 23 52  12 0.48 ± 0.07 0.56 ± 0.03 21 ± 5  22  12t 1.1 ± 0.2 0.78 ± 0.04 180 ± 160 53  40 0.19 ± 0.09 0.19 ± 0.04 10 ± 1  28  40t^(f) 20 ± 1  — — 54  84 0.82 ± 0.2  0.45 ± 0.06 21 ± 5  36  84t 1.8 ± 0.4 0.53 ± 0.07 31 ± 10 55 126 0.14 ± 0.04 0.40 ± 0.04 11 ± 3  38 126t 1.4 ± 0.2 0.54 ± 0.03 181 ± 57  56 ^(a)Binding experiments were done as described in Example 2 and errors are given as standard deviations. ^(b)Full length, and truncated ligands are listed in pairs and represent sequence families 1-6, in order. ^(c)Dissociation constant of the higher-affinity binding component as defined in eq. 5. ^(d)Mole fraction of the high-affinity binding component as defined in eq. 5. ^(e)Dissociation constant of the lower-affinity binding component as defined in eq. 5. #^(d)Mole fraction of the high-affinity binding component as defined in eq. 5. ^(e)Dissociation constant of the lower-affinity binding component as defined in eq. 5. ^(f)Dissociation constant for ligand 40t was determined by fitting the data points to eq. 2.

TABLE 4 VEGF VEGF SEQ Protein Receptor ID Binding Binding NO: Ligand SEQUENCE Kd Ki 147 NX-107 ACC CUG AUG GUA GAC GCC GGG GUG 1 nM 148 NX-176 ACC CUG AUG GUA GAC GCC GGG GUG  65 nM 10 nM  149 NX-178 T*T*T*T* ACC CUG AUG GUA GAC GCC GGG GUG T*T*T*T*T 0.7 nM 1 nM 150 NX-190 T*T*T*T* ACC CTG ATG GTA GAC GTT GGG GTG T*T*T*T*T 151 NX-191 T*T*T*T* ACC CUG AUG GUA GAC GCC GGG GUG T*T*T*T*T 120 nM 500 nM   152 NX-213 T*T*T*T* ACC CUG AUG GUA GAC GCC GGG GUG T*T*T*T*T 0.2 nM 1 nM 153 NX-214 T*T*T*T* ACC CUG AUG GUA GAC GCC GGG GUG T*T*T*T*T 0.2 nM 1 nM 154 NX-215 T*T*T*T* ACC CUG A*UG GUA GAC GCC GGG GUG T*T*T*T*T 0.2 nM 1 nM 155 NX-203 ACC CUG AUG GUA GAC GCC GGG GUG 156 NX-204 ACC CUG AUG GUA GAC GCC GGG GUG 157 NX-205 ACC CUG AUG GUA GAC GCC GGG GUG 158 NX-206 ACC CUG AUG GUA GAC GCC GGG GUG N = 2′OH N = 2′NH₂ N = 2′OMe N* = phosphorothioate N = 2′deoxy N = 2′OMe:2′OH::2:1

TABLE 8 VEGF ssDNA ligands ssDNA bulk pool, BH SELEX:0.44 nM SEQ ID NO: ligand Family 1 Kd, nM 159 3         acaacggcgtggaagactagagtgcagccgaacgcatcta 160 5     acgctacaagtccgctgtggtagacaagagtgcaggcaag 161 9 (3x)             aggcccgtcgaagntagagcgcagggccccaaaataccg 162 10      gtaccatccacggtttacgtggacaagagggccctggtac 1 163 11     tcactacaagtccgccgtggtagacaagagtgcaggcaag 164 15      accgctgtgtagttcctttaggactagagggccgcctac 0.88 165 21       taggcttgacgtcttctgactagagtgcagtcaaaccc 166 27           tgcaggtcgactctagaggatccccgggtaccgagctcga 167 31          acggtttacgtggacaagagggccctggtac 168 32 (3x)                 ggtggactagaggncagcaaacgatccttggttcgcgtcc 2 169 33     tcaagcactcccgtcttccagacaagagtgcagggcctct 2 170 35            cgtgatggacaagagggccctatccaccggatatccgtc 171 37     caagcagtgcccgtcttccagacaagagtgcaggcctct 172 39 tgatccaccgtttatagtccgtggtagacaagagtgcagg 173 41               aacacacaagaggacagttacaggtaacatccgctcagg 174 49          agtggcgtctatagacaagagtgcagcccgagtttca 175 50                 ccacaagagggcagcaagtg-tacaactacagcgtccgg 176 b56(8x)         gcagggccacgtctattagactagagtgcagtggttc 0.5 177 b69     acggtccaaaggtttcccatccgtggactagagggcacgtgctta 178 b80    ccgtcgcgtgactataaccacacgcagactagagtgcagggctta 8.1 179 b81**            ccgaatggggctgcgactgcagtggacgtcacgtcgtta 0.3 180 b91**                acgcaagagagtcnccgaatgcagtctcagccgctaaca 231 Consensus                   agacaagagtgcagg 232                     ggactagagggcagt Family 2 Kd PCR 181 2       cannncactgcaagcaattgtggcccaaagggctgagt 182 14          gctcgcttacaaaagggagccactgtagcccagactggac 183 25 (2x)       ggttatggtgtggttccgaatggtgggcaaagtaacgctt 184 40        gcttgngctccgaaggggcgcgtatccaaggacggttc 185 46       tatggagtggttccgaatggtgggcaaagtaacgctt 186 b54     tgcnngcgggcggttctccggatgggaccataaggctttagctta 2.5 187 b55       acaaggggtcctgnngaatgggggaatacgctagccgaa 15 188 b59 aacacgagcatgtggggtcccttccgaatggggggtacaggctta 91 189 b79       gaggcattaggtccgaatggtagtaatgctgtcgtgccttgctta 2 190 b81**                ccgaatggggctgcgactgcagtggacgtcacgtcgtta 0.3 191 b85      gaggaggtgcgttgtccgaaggggtcgttagtcacctcgtgctta .15 192 b88 (5x)        gcaaggggtcctgccgaatgggggaatacgctagccgaaa 34 193 b89 (3x)            atccttccgaatgggggaaatggcgnccca 2-3 194 b91**       acgcaagagaggtcnccgaatggcagtctcagccgctaaca 3.9 195 b99        cacgataatcctccgaaagcgttgtccgaatgggtcgttagctta 34 Consensus                 ctccgaatgggggnaaa g Family 3 196 18  tatcacccccactggatagagccgcagcgtgcccctact 197 19      gcccactgcatagagggacggttgtttccgcccggtgttt 198 b51 gtgaaggagccccaactggatagaagccttaaggcggtgt 199 b60        ccaccgcagagtgttacaccccataggagaagtccggatggctta 26 200 b62        ccactgcatagagagtcgcaagacacggtgctttattcnccgctta 2.9 201 b63      tgccccactggatagagtaggaggcctagccgacacggtgctta 202 b65   cgaggtcccccactggatagagttgttgaaacaacggtgcgctta 0.53 203 b66   aacacttccccactggatagaggcctttcgcagagccggtgctta 1.3 204 b95         ccactgcatagagaactggatcgacggtccaaagttcggtgctta 0.9 205 b96         ccactgcatagagatactggattcgacnnnccaaagtttcggtgctta 1.5 206 b97         ccactgcagagagtcaaccttacgangccaaggttgcggtgctta >1 234 Consensus           ccccactggatagag 235           ccccactgcatagag Family 4 207 1    tctgcgagagacctactggaacgttttgtgatattcaca 12 208 6  atacacccggcgggcctaccggatcgttgatttctctcc 1.0 209 13   acgccccctgagacctaccggaatnttntcgctcgctaggccta 210 23 gggcatctaacccagacctaccggaacgttatcgcttgtg 0.75 211 44   ggtgtgaaccagacctacnggaacgttatcgcttgtg 0.4 236 Consensus                agacctaccggaacgtt Orphans 212 4   catcagtattatataacgggaaccaacggcaaatgctgac 213 7 tccnngggagaatagggttagtcggagaagttaatcgct 214 16   cgggaacgtgtggttacncggcctactggattgtttcctg 215 30   ggtaggtccggtgtgaaagaggttcgcatcaggta 216 38  cctcaggcaacatagttgagcatcgtatcgatcctggag 217 43  ttggcttgagtcccgggacgcactgttgacagtggagt 218 45   cagcaggttagtataacgggaaccaacggcaaatgctgac 219 b53    gcaagggcatctccggaatcggttaatctgacttgcaatacgctta 2.5 220 b98    gatccacgaagaagcttactctcatgtagttcca >100 Truncates 221 10t    gtaccatccacggtttacgtggacaagagggccctggtac 5 222 15t         gtagttcctttaggactagagggccgcctac 3 223 32t               tggactagaggncagcaaacgatccttggttcgcgtcc 17 224 33t          cccgtcttccagacaagagtgcaggg 0.7 225b 56t       agggccacgtctatttagactagttagtgcagtggttc 0.2 226b 85t        ggaggtgcgttgtccgaaggggtcgagtcacctc 0.3 227 88t        gcaaggggtcctgccgaatgggggaatacgctagccgaaa 19 228b 65t cgaggtcccccactggatagagttgttgaaacaacggtgcgctta 0.32 229b 66t aacacttccccactggatagaggcctttcgcagagccggtgctta 0.35 230b 23t            gggcatctaacccagacctaccggaacgttatcgcttgtg >200

242 77 base pairs nucleic acid single linear 1 GGGAGCUCAG AAUAAACGCU CAANNNNNNN NNNNNNNNNN NNNNNNNNNN 50 NNNUUCGACA UGAGGCCCGG AUCCGGC 77 48 base pairs nucleic acid single linear 2 CCGAAGCTTA ATACGACTCA CTATAGGGAG CTCAGAATAA ACGCTCAA 48 24 base pairs nucleic acid single linear 3 GCCGGATCCG GGCCTCATGT CGAA 24 77 base pairs nucleic acid single linear 4 GGGAGCUCAG AAUAAACGCU CAAGAGUGAU GCUCAUCCGC ACUUGGUGAC 50 GUUUUCGACA UGAGGCCCGG AUCCGGC 77 77 base pairs nucleic acid single linear 5 GGGAGCUCAG AAUAAACGCU CAAUACCGGC AUGCAUGUCC AUCGCUAGCG 50 GUAUUCGACA UGAGGCCCGG AUCCGGC 77 77 base pairs nucleic acid single linear 6 GGGAGCUCAG AAUAAACGCU CAAUGCGUGU UGUGACGCAC AUCCGCACGC 50 GCAUUCGACA UGAGGCCCGG AUCCGGC 77 77 base pairs nucleic acid single linear 7 GGGAGCUCAG AAUAAACGCU CAAGGAGUGA UGCCCUAUCC GCACCUUGGC 50 CCAUUCGACA UGAGGCCCGG AUCCGGC 77 77 base pairs nucleic acid single linear 8 GGGAGCUCAG AAUAAACGCU CAAGCUUGAC NGCCCAUCCG AGCUUGAUCA 50 CGCUUCGACA UGAGGCCCGG AUCCGGC 77 77 base pairs nucleic acid single linear 9 GGGAGCUCAG AAUAAACGCU CAAUCCUUGA UGCGGAUCCG AGGAUGGGAC 50 GUUUUCGACA UGAGGCCCGG AUCCGGC 77 77 base pairs nucleic acid single linear 10 GGGAGCUCAG AAUAAACGCU CAAACACCGU CGACCUAUGA UGCGCAUCCG 50 CACUUCGACA UGAGGCCCGG AUCCGGC 77 76 base pairs nucleic acid single linear 11 GGGAGCUCAG AAUAAACGCU CAACCGGUAG UCGCAUGGCC CAUCGCGCCC 50 GGUUCGACAU GAGGCCCGGA UCCGGC 76 77 base pairs nucleic acid single linear 12 GGGAGCUCAG AAUAAACGCU CAAGUCAGCA UGGCCCACCG CGCUUGACGU 50 CUGUUCGACA UGAGGCCCGG AUCCGGC 77 77 base pairs nucleic acid single linear 13 GGGAGCUCAG AAUAAACGCU CAACACGGUU CGAUCUGUGA CGUUCAUCCG 50 CACUUCGACA UGAGGCCCGG AUCCGGC 77 77 base pairs nucleic acid single linear 14 GGGAGCUCAG AAUAAACGCU CAAGGAGCAG UGACGCACAU CCACACUCCA 50 GCGUUCGACA UGAGGCCCGG AUCCGGC 77 77 base pairs nucleic acid single linear 15 GGGAGCUCAG AAUAAACGCU CAAUUCGAAU GCCGAGGCUC GUGCCUUGAC 50 GGGUUCGACA UGAGGCCCGG AUCCGGC 77 77 base pairs nucleic acid single linear 16 GGGAGCUCAG AAUAAACGCU CAAUCGCGAA UGCCGACCAC UCAGGUUGAU 50 GGGUUCGACA UGAGGCCCGG AUCCGGC 77 77 base pairs nucleic acid single linear 17 GGGAGCUCAG AAUAAACGCU CAAUGCCGGC CUGAUCGGCU GAUGGGUUGA 50 CCGUUCGACA UGAGGCCCGG AUCCGGC 77 77 base pairs nucleic acid single linear 18 GGGAGCUCAG AAUAAACGCU CAAGAAUGCC GAGCCCUAAG AGGCUUGAUG 50 UGGUUCGACA UGAGGCCCGG AUCCGGC 77 77 base pairs nucleic acid single linear 19 GGGAGCUCAG AAUAAACGCU CAACCUUNAU GUGGCNCGAA CUGCGUGCCG 50 AGGUUCGACA UGAGGCCCGG AUCCGGC 77 77 base pairs nucleic acid single linear 20 GGGAGCUCAG AAUAAACGCU CAAGCUUGAU GGGUGACACA CGUCAUGCCG 50 AGCUUCGACA UGAGGCCCGG AUCCGGC 77 77 base pairs nucleic acid single linear 21 GGGAGCUCAG AAUAAACGCU CAAGUCGUCC UGCAUGGGCC GUAUCGGUGC 50 GCGUUCGACA UGAGGCCCGG AUCCGGC 77 77 base pairs nucleic acid single linear 22 GGGAGCUCAG AAUAAACGCU CAAGCAGACG AAGGGAACCU GCGUCUCGGC 50 ACCUUCGACA UGAGGCCCGG AUCCGGC 77 77 base pairs nucleic acid single linear 23 GGGAGCUCAG AAUAAACGCU CAAAAGGAGG ANCCUGCGUC UCGGCACUCC 50 GCAUUCGACA UGAGGCCCGG AUCCGGC 77 77 base pairs nucleic acid single linear 24 GGGAGCUCAG AAUAAACGCU CAAGGGAACC UGCGUUUCGG CACCUUGUUC 50 CGUUUCGACA UGAGGCCCGG AUCCGGC 77 79 base pairs nucleic acid single linear 25 GGGAGCUCAG AAUAAACGCU CAAAAAUGUG GGUUACCUGC GUUUCGGCAC 50 CACGUUUCGA CAUGAGGCCC GGAUCCGGC 79 77 base pairs nucleic acid single linear 26 GGGAGCUCAG AAUAAACGCU CAACGACGGU AGAGUCUGUC CCGUCAUCCC 50 CCAUUCGACA UGAGGCCCGG AUCCGGC 77 77 base pairs nucleic acid single linear 27 GGGAGCUCAG AAUAAACGCU CAAAAAGACC CCUGGUUGAG UCUGUCCCAG 50 CCGUUCGACA UGAGGCCCGG AUCCGGC 77 77 base pairs nucleic acid single linear 28 GGGAGCUCAG AAUAAACGCU CAAGACCCAU CGUCAACGGU UGAGUCUGUC 50 CCGUUCGACA UGAGGCCCGG AUCCGGC 77 77 base pairs nucleic acid single linear 29 GGGAGCUCAG AAUAAACGCU CAAGGUUGAG UCUGUCCCUU CGAGUAUCUG 50 AUCUUCGACA UGAGGCCCGG AUCCGGC 77 77 base pairs nucleic acid single linear 30 GGGAGCUCAG AAUAAACGCU CAAUCGGACA GUUGGUUGAG UCUGUCCCAA 50 CUUUUCGACA UGAGGCCCGG AUCCGGC 77 76 base pairs nucleic acid single linear 31 GGGAGCUCAG AAUAAACGCU CAAGACCAUG UGACUGGUUG AGCCUGUCCC 50 AGUUCGACAU GAGGCCCGGA UCCGGC 76 77 base pairs nucleic acid single linear 32 GGGAGCUCAG AAUAAACGCU CAAAACGGUU GAGUCUGUCC CGUAAGAGAG 50 CGCUUCGACA UGAGGCCCGG AUCCGGC 77 77 base pairs nucleic acid single linear 33 GGGAGCUCAG AAUAAACGCU CAAUCGGAAU GUAGUUGACG UAUCCUUGUC 50 CGAUUCGACA UGAGGCCCGG AUCCGGC 77 77 base pairs nucleic acid single linear 34 GGGAGCUCAG AAUAAACGCU CAAGGGUGUA GUUGGGACCU AGUCCGCCGU 50 ACCUUCGACA UGAGGCCCGG AUCCGGC 77 77 base pairs nucleic acid single linear 35 GGGAGCUCAG AAUAAACGCU CAAGGCAUAG UUGGGACCUC GUCCGCCGUG 50 CCCUUCGACA UGAGGCCCGG AUCCGGC 77 77 base pairs nucleic acid single linear 36 GGGAGCUCAG AAUAAACGCU CAAUAGUUGG AGGCCUGUCC UCGCCGUAGA 50 GCGUUCGACA UGAGGCCCGG AUCCGGC 77 77 base pairs nucleic acid single linear 37 GGGAGCUCAG AAUAAACGCU CAAGGGGUUC UAGUGGAGAC UCUGCCGCGG 50 CCCUUCGACA UGAGGCCCGG AUCCGGC 77 77 base pairs nucleic acid single linear 38 GGGAGCUCAG AAUAAACGCU CAAACGGUUC UGUGUGUGGA CUAGCCGCGG 50 CCGUUCGACA UGAGGCCCGG AUCCGGC 77 77 base pairs nucleic acid single linear 39 GGGAGCUCAG AAUAAACGCU CAAGGGAUGU UUGGCUAUCU CGGAUAGUGC 50 CCCUUCGACA UGAGGCCCGG AUCCGGC 77 77 base pairs nucleic acid single linear 40 GGGAGCUCAG AAUAAACGCU CAAGCUUAAU ACGACUCACU NUAGGGAGCU 50 CAGUUCGACA UGAGGCCCGG AUCCGGC 77 77 base pairs nucleic acid single linear 41 GGGAGCUCAG AAUAAACGCU CAAUUGAGUG AUGUGCUUGA CGUAUCGCUG 50 CACUUCGACA UGAGGCCCGG AUCCGGC 77 15 base pairs nucleic acid single linear RNA N 15 This symbol stands for the complimentary base for the N located in position 1 42 NUGAUGVNCA UCCGN 15 22 base pairs nucleic acid single linear RNA S 11 and 12 This symbol stands for the complimentary base for the S located in positions 9 and 10 43 AAUGCCGASS SSUUGAUGGG UU 22 25 base pairs nucleic acid single linear RNA H 24 This symbol stands for the complimentary base for the D located in position 2 Y 25 This symbol stands for the complimentary base for the R located in position 25 44 RDGGGAACCU GCGUYUCGGC ACCHY 25 19 base pairs nucleic acid single linear RNA D 18 and 19 This symbol stands for the complimentary base for the H located in positions 1 and 2 45 HHGGUUGAGU CUGUCCCDD 19 27 base pairs nucleic acid single linear RNA N 18-20 and 27 This symbol stands for the complimentary base for the N located in positions 1 and 10-12 46 NRUAGUUGGN NNCUNSUNNN CGCCGUN 27 32 base pairs nucleic acid single linear RNA M 20 This symbol stands for the complimentary base for the K located in position 14 S 31 This symbol stands for the complimentary base for the S located in position 2 47 RSGGUUUCRU GUGKRGACUM UGCCGCGGCC SU 32 21 base pairs nucleic acid single linear 48 GGGAACCUGC GUYUCGGCAC C 21 15 base pairs nucleic acid single linear 49 GGUUGAGUCU GUCCC 15 40 base pairs nucleic acid single linear 50 AAGCAGACGA AGGGAACCUG CGUCUCGGCA CCUUCGACAU 40 31 base pairs nucleic acid single linear 51 GGCCGGUAGU CGCAUGGCCC AUCGCGCCCG G 31 35 base pairs nucleic acid single linear 52 GGAAGCUUGA UGGGUGACAC ACGUCAUGCC GAGCU 35 29 base pairs nucleic acid single linear 53 GGAAGGGAAC CUGCGUCUCG GCACCUUCG 29 29 base pairs nucleic acid single linear 54 GGUCAACGGU UGAGUCUGUC CCGUUCGAC 29 36 base pairs nucleic acid single linear 55 GGCUCAAUAG UUGGAGGCCU GUCCUCGCCG UAGAGC 36 35 base pairs nucleic acid single linear 56 GGAACGGUUC UGUGUGUGGA CUAGCCGCGG CCGUU 35 76 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 57 GGGAGACAAG AAUAACGCUC AANNNNNNNN NNNNNNNNNN NNNNNNNNNN 50 NNUUCGACAG GAGGCUCACA ACAGGC 76 39 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 58 TAATACGACT CACTATAGGG AGACAAGAAU AACGCUCAA 39 24 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 59 GCCTGTTGTG AGCCTCCTGT CGAA 24 81 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 60 GGGAGGACGA UGCGGNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 50 NNNNNNNNNN NNNNNCAGAC GACTCGCCCG A 81 32 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 61 TAATACGACT CACTATAGGG AGGACGAUGC GG 32 16 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 62 TCGGGCGAGT CGTCTG 16 81 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 63 GGGAGGACGA UGCGGUGGCU GUGAUCAAUG CGGGGAGGUG AGGAAGGGCC 50 UUACAAAUCC UUCGGCAGAC GACTCGCCCG A 81 81 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 64 GGGAGGACGA UGCGGUGUGA UCAAUGCGGU GGCGGGGUAU GGAUGGGAGU 50 CUGGAAUGCU GCGCUCAGAC GACTCGCCCG A 81 80 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 65 GGGAGGACGA UGCGGCGCUG UGUUCAAUGC GGGGAUCGGG CCGGAGGAUG 50 AACAAAUGGC GGGUCAGACG ACTCGCCCGA 80 81 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 66 GGGAGGACGA UGCGGUGUUG AGCAAGCACU CAUGUGGUCA AUGUGGGAGU 50 GGGAGCUGGU GGGGUCAGAC GACTCGCCCG A 81 81 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 67 GGGAGGACGA UGCGGCAAGG GAGCGUUAGA GCCAUGUGGU CAAUGAGGGG 50 UGGGAUUGGU UGGGUCAGAC GACTCGCCCG A 81 81 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 68 GGGAGGACGA UGCGGCAUGG UUGUGAACUG UUGUGAUCAA UGCGGGGAGG 50 GUAAUGGUGG GUGGUCAGAC GACTCGCCCG A 81 81 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 69 GGGAGGACGA UGCGGAUGAG UGACACAUGU GCUCAAUGCG GGGUGGGUUG 50 GUAGGGGUAG CACGGCAGAC GACTCGCCCG A 81 80 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 70 GGGAGGACGA UGCGGUGUGG UCAAUGUGGG GUAGGGCUGG UAGGGCAUUC 50 CGUACUGGUG UGGUCAGACG ACTCGCCCGA 80 80 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 71 GGGAGGACGA UGCGGCCGAG UUGUGCUCAA UGUGGGGUCU GGGUACGGAC 50 GGGAACAGAU CUGGCAGACG ACTCGCCCGA 80 79 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 72 GGGAGGACGA UGCGGGUGCU CAGCAUUGUG UGCUCAAUGC GGGGGAGUUU 50 GGGUUGGCGA CGGCAGACGA CTCGCCCGA 79 16 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 73 UGUGNUCAAU GNGGGG 16 81 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 74 GGGAGGACGA UGCGGCAUAG GCUUACAACA GAGCGGGGGU UCUGAUGGUA 50 GACGCCGGGA CGCCCCAGAC GACTCGCCCG A 81 80 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 75 GGGAGGACGA UGCGGUAUGA UGGUAGACGC CGUACCGCAU CAGGCCAAGU 50 CGUCACAGAU CGUGCAGACG ACTCGCCCGA 80 76 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 76 GGGAGACAAG AAUAACGCUC AAGCAACAGA GGCUGAUGGU AGACGCCGGC 50 CAUUCGACAG GAGGCUCACA ACAGGC 76 76 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 77 GGGAGACAAG AAUAACGCUC AAAGAGUCGC UGAUGGUAGA CGCCGGCGGA 50 UCUUCGACAG GAGGCUCACA ACAGGC 76 75 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 78 GGGAGACAAG AAUAACGCUC AAGAGGCUGA UGGCAGACGC GGCCGAAGAC 50 AUUCGACAGG AGGCUCACAA CAGGC 75 75 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 79 GGGAGACAAG AAUAACGCUC AACCCUGAUG GUAGACGCCG GGGUGCCGGA 50 AUUCGACAGG AGGCUCACAA CAGGC 75 17 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 80 CUGAUGGUAG ACGCCGG 17 82 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 81 GGGAGGACGA UGCGGCAGUG CUGAACUAAU CGAACGGGGU CAAGGAGGGU 50 CGAACGAGAU CUGCCGCAGA CGACTCGCCC GA 82 81 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 82 GGGAGGACGA UGCGGCACCU UCGUGGGGUC AAGGAGGGUC GCGAGGCCGC 50 AGGAUCAACC GUGUGCAGAC GACTCGCCCG A 81 81 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 83 GGGAGGACGA UGCGGGGUCA AGUUGGGUCG AGGAAGCGCU CCCGAGUAUC 50 GUAGUGUGCG ACUGCCAGAC GACTCGCCCG A 81 75 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 84 GGGAGACAAG AAUAACGCUC AAGAACUUGA UCGGGGUCAA GGCGGGACGA 50 AUUCGACAGG AGGCUCACAA CAGGC 75 76 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 85 GGGAGACAAG AAUAACGCUC AAUGGCGGGA CCAAGGAGGG ACGUGUAGGA 50 AAUUCGACAG GAGGCUCACA ACAGGC 76 78 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 86 GGGAGACAAG AAUAACGCUC AAAAAAUGCA CAAAUCGGGG UCAAGGAGGG 50 ACGAUUCGAC AGGAGGCUCA CAACAGGC 78 78 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 87 GGGAGGACGA UGCGGAUGGG UUCGUGUGGU GAAUGGAGGA GGUGGGCUCG 50 CAUGCUACUG UGCAGACGAC TCGCCCGA 78 12 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 88 GGUCAAGGNG GG 12 75 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 89 GGGAGGACGA UGCGGUGCAC UAAGUCCGGG UAGUGGGAGU GGUUGGGCCU 50 GGAGUGCGCC AGACGACTCG CCCGA 75 76 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 90 GGGAGACAAG AAUAACGCUC AAAUCAAAGG GUAGAGGGUG GGCUGUGGCA 50 AGUUCGACAG GAGGCUCACA ACAGGC 76 76 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 91 GGGAGACAAG AAUAACGCUC AAAAUCGAGG GUAGCGGGCG CGGCUUGGCC 50 AAUUCGACAG GAGGCUCACA ACAGGC 76 77 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 92 GGGAGACAAG AAUAACGCUC AAGCCUCGGA UCGGGCAGCG GGUGGGAUGG 50 CAAUUCGACA GGAGGCUCAC AACAGGC 77 76 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 93 GGGAGACAAG AAUAACGCUC AAAACGGAGU GGUAGGCGUU GGGUGCCAGG 50 AAUUCGACAG GAGGCUCACA ACAGGC 76 11 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 94 GGUAGNGGGN G 11 79 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 95 GGGAGGACGA UGCGGAACCG AGUCGUGUGG GUUGGGGCUC CAGUACAUCC 50 CCGGUCUGGG UGUCAGACGA CTCGCCCGA 79 79 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 96 GGGAGGACGA UGCGGUAACA UACGCAGUCG UGUGGGUAGG GGAUCACAAA 50 CUGCGUAUCG UGUCAGACGA CTCGCCCGA 79 65 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 97 GGGAGACAAG AAUAACGCUC AAAGUCGUGU GGGUGGGGUC AUUCGACAGG 50 AGGCUCACAA CAGGC 65 76 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 98 GGGAGACAAG AAUAACGCUC AAAGUGUAGG AUAGGGGAUG GGAGGUCCGG 50 GAUUCGACAG GAGGCUCACA ACAGGC 76 76 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 99 GGGAGACAAG AAUAACGCUC AAACUGUGGG CUCUAGGGCA GUGGGAGUGG 50 AGUUCGACAG GAGGCUCACA ACAGGC 76 76 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 100 GGGAGACAAG AAUAACGCUC AAAGUGGGAC AGGGAUUGCG GAGGGUGGAA 50 GGUUCGACAG GAGGCUCACA ACAGGC 76 76 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 101 GGGAGACAAG AAUAACGCUC AAGUCAGGAG GACUGGAAGG UGGGACUGGU 50 GAUUCGACAG GAGGCUCACA ACAGGC 76 76 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 102 GGGAGACAAG AAUAACGCUC AAGCAGGAGA GAGGGUGUUG GGUGCGGAUA 50 CAUUCGACAG GAGGCUCACA ACAGGC 76 80 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 103 GGGAGGACGA UGCGGAGGGU AGGAGGCUAA GCAUAGUUCA GAGGAGGUGG 50 CGCGUGCCCC CGUGCAGACG ACTCGCCCGA 80 80 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 104 GGGAGGACGA UGCGGCAACA UUGGCACCAA UGCUCUGUGU UAAUGUGGGG 50 UGGGAACGGC GCCGCAGACG ACTCGCCCGA 80 79 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 105 GGGAGGACGA UGCGGACCAA UGAUUGCAAU GAGGGCAGUG GGGGGGAAUU 50 GGGCUCGUGU GGUCAGACGA CTCGCCCGA 79 81 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 106 GGGAGGACGA UGCGGGCAGU GGGUGAGGUC CGGGCACGAU UGAGUUUGAA 50 CGGUUCUGGC UUGGUCAGAC GACTCGCCCG A 81 81 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 107 GGGAGGACGA UGCGGGUGGU AGGUGUAGAG UGGAUGGCGG AGGUCCUAGU 50 AGUUCUGUGC CUGGUCAGAC GACTCGCCCG A 81 72 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 108 GGGAGGACGA UGCGGCGCGG GAGAGGGUAG UGGGUGUGGU GCUUGGACAA 50 GCAGCGCAGA CGACTCGCCC GA 72 81 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 109 GGGAGGACGA UGCGGACCCG CAUACGGACC GCGGAGGGGG AAAUCUAGCC 50 UCAGGGGUGG CGGGCCAGAC GACTCGCCCG A 81 80 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 110 GGGAGGACGA UGCGGUGAAG AAGCGGGGAC UGCACGACGG GAUGGAGGGA 50 CACGACUGCG GGGUCAGACG ACTCGCCCGA 80 76 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 111 GGGAGACAAG AAUAACGCUC AAACACCAGG AGAGUGGGUU CGGGUGAGGA 50 CGUUCGACAG GAGGCUCACA ACAGGC 76 76 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 112 GGGAGACAAG AAUAACGCUC AAGUGGCUGA UGGCAGACGC CGGCUGCUGA 50 CGUUCGACAG GAGGCUCACA ACAGGC 76 76 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 113 GGGAGACAAG AAUAACGCUC AAUCGUGCCA GGACAUGGUG GCUCAUGGGU 50 AAUUCGACAG GAGGCUCACA ACAGGC 76 76 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 114 GGGAGACAAG AAUAACGCUC AAAGGUACGG GGGAGGGAAG GAUAUAACGC 50 GAUUCGACAG GAGGCUCACA ACAGGC 76 76 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 115 GGGAGACAAG AAUAACGCUC AAUGGAAAGG UGUGGAAAGA GGCAUCGGGG 50 GGUUCGACAG GAGGCUCACA ACAGGC 76 76 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 116 GGGAGACAAG AAUAACGCUC AAUCAAUGGG CAGGAAGAGG GAAGGGAUGU 50 GAUUCGACAG GAGGCUCACA ACAGGC 76 76 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 117 GGGAGACAAG AAUAACGCUC AACAUGGGUA AGGGAGUGGG AGUGGUGAAU 50 AGUUCGACAG GAGGCUCACA ACAGGC 76 76 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 118 GGGAGACAAG AAUAACGCUC AAGGAACGAG UAGGGCAGUG GGUGGUGAUG 50 GCUUCGACAG GAGGCUCACA ACAGGC 76 76 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 119 GGGAGACAAG AAUAACGCUC AAUAGGGCAG AGGGAGUGGU UAGGGCUGUG 50 AUUUCGACAG GAGGCUCACA ACAGGC 76 76 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 120 GGGAGACAAG AAUAACGCUC AAGGGUAGUG GGAAGGGUAA GGGCCGAGGU 50 GGUUCGACAG GAGGCUCACA ACAGGC 76 76 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 121 GGGAGACAAG AAUAACGCUC AAAAUACACA CCGCGGGAAG GGAGGGUGGA 50 AAUUCGACAG GAGGCUCACA ACAGGC 76 76 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 122 GGGAGACAAG AAUAACGCUC AAAGACUACA GCGCGGGUUA GGGUUGAGGG 50 AAUUCGACAG GAGGCUCACA ACAGGC 76 76 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 123 GGGAGACAAG AAUAACGCUC AAUACGAGCA AGCGGGCGAA GGGUUGAGGG 50 AAUUCGACAG GAGGCUCACA ACAGGC 76 76 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 124 GGGAGACAAG AAUAACGCUC AACAAGGUGG UGGAGGAGGA UACGAUCUGC 50 AGUUCGACAG GAGGCUCACA ACAGGC 76 75 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 125 GGGAGACAAG AAUAACGCUC AAGGAGGGAA GGAGGGCAGG UGAUGGGUCA 50 GUUCGACAGG AGGCUCACAA CAGGC 75 76 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 126 GGGAGACAAG AAUAACGCUC AAUGAUGGCG GUAGUGGAGG UAAUGAGCGU 50 GAUUCGACAG GAGGCUCACA ACAGGC 76 72 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 127 GGGAGACAAG AAUAACGCUC AAGCAACUGG GGGCGGGUGG UGUGAGGAUU 50 CGACAGGAGG CUCACAACAG GC 72 76 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 128 GGGAGACAAG AAUAACGCUC AAGGAGGGGC CUAUAGGGGU GGUGGUGUAC 50 GAUUCGACAG GAGGCUCACA ACAGGC 76 76 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 129 GGGAGACAAG AAUAACGCUC AAUAUAGGGU AGUGGGUGUA GGUAGGGCGA 50 CAUUCGACAG GAGGCUCACA ACAGGC 76 76 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 130 GGGAGACAAG AAUAACGCUC AAGAGGGUUG GAGGGCAUGG GGCAGGAACC 50 GGUUCGACAG GAGGCUCACA ACAGGC 76 76 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 131 GGGAGACAAG AAUAACGCUC AACGUAGAAC UGGCGGGCAG UGGGGGGGAU 50 GCUUCGACAG GAGGCUCACA ACAGGC 76 76 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 132 GGGAGACAAG AAUAACGCUC AAUGAGGGGA CGAGGGAUGU GGGGAGCGGG 50 ACUUCGACAG GAGGCUCACA ACAGGC 76 76 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 133 GGGAGACAAG AAUAACGCUC AACGAGGGAU GGGAGGCGUG UGGAAGAUGC 50 AAUUCGACAG GAGGCUCACA ACAGGC 76 76 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 134 GGGAGACAAG AAUAACGCUC AAGCAUCCGG GGACAAGAUG GGUCGGUAAG 50 GUUUCGACAG GAGGCUCACA ACAGGC 76 75 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 135 GGGAGACAAG AAUAACGCUC AAGUGUGCGG GGUCAAGACG GGUGGCGUGC 50 GUUCGACAGG AGGCUCACAA CAGGC 75 76 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 136 GGGAGACAAG AAUAACGCUC AAUCAAACCA UGGGGCGGGU GGUACGAGGA 50 ACUUCGACAG GAGGCUCACA ACAGGC 76 76 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 137 GGGAGACAAG AAUAACGCUC AACGAGUCCG AGGGAUGGGU GGUGUGCGGC 50 AAUUCGACAG GAGGCUCACA ACAGGC 76 76 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 138 GGGAGACAAG AAUAACGCUC AACAGUGUCG GAGAGGAGGA UGGAGGUAUG 50 AAUUCGACAG GAGGCUCACA ACAGGC 76 81 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 139 GGGAGGACGA UGCGGCACCA CUACGCGGGA AGGGUAGGGU GGAUUACAAG 50 GUGUGACCGC UCCGUCAGAC GACTCGCCCG A 81 80 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 140 GGGAGGACGA UGCGGUACGG UUAACGGGGG UGGUGUGGGA GGACACAAAG 50 CGCGUACCUG CCCCCAGACG ACTCGCCCGA 80 81 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 141 GGGAGGACGA UGCGGAGGUC CUCGAGGGUC UGGGUGUGGG AGUGGGCAUG 50 GACCAAUACC GCGUGCAGAC GACTCGCCCG A 81 81 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 142 GGGAGGACGA UGCGGAAACC CAUCCUGCGC GGGAUGGGAG GGUGGAAACA 50 CUAGAGCUUC GCCUGCAGAC GACTCGCCCG A 81 81 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 143 GGGAGGACGA UGCGGAACUG GUGGUCACGC GUUGAGGUGG UGGAGGUGGA 50 UUCAACGGUC GAGGGCAGAC GACTCGCCCG A 81 81 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 144 GGGAGGACGA UGCGGCAUGA AAGUAGGGUU AUGAAGGCGG UAGAUGGAGG 50 AGGUUGGGUU GCCGCCAGAC GACTCGCCCG A 81 81 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 145 GGGAGGACGA UGCGGGUCUA UUGGGUAGGU GUUUGCAAGA AUUCCGCACG 50 AUAGGUAAAA CAGUGCAGAC GACTCGCCCG A 81 80 base pairs nucleic acid single linear All C′S are 2′-NH2 cytosine All U′S are 2′-NH2 uracil 146 GGGAGGACGA UGCGGUGUAG GGGAAGUACG AGAGUGGGAG CGGCCGUAUA 50 GGUGGGAGUG UGCUCAGACG ACTCGCCCGA 80 24 base pairs nucleic acid single linear modified base 2..4, 15, 17..18 C at positions 2-4, 15, 17-18 are 2′NH2 cytosine modified base 5, 8, 11, 23 U at positions 5, 8, 11, 23 are 2′NH2 uracil 147 ACCCUGAUGG UAGACGCCGG GGUG 24 24 base pairs nucleic acid single linear modified base 2..4, 15, 17..18 C at positions 2-4, 15, 17-18 are 2′NH2 cytosine modified base 5, 8, 11, 23 U at positions 5, 8, 11 and 23 are 2′NH2 uracil modified base 12, 14 A at positions 12 and 14 are 2′OMe adenosine modified base 13, 16, 19..21, 22, 24 G at positions 13, 16, 19-21, 22 and 24 are 2′OMe guanosine 148 ACCCUGAUGG UAGACGCCGG GGUG 24 33 base pairs nucleic acid single linear modified base 6..8, 19, 21..22 C at positions 6-8, 19 and 21-22 are 2′NH2 cytosine modified base 9, 12, 15, 27 U at positions 9, 12, 15 and 27 are 2′NH2 uracil modified base 1..4, 29..33 T at positions 1-4 and 29-33 are 2′ deoxy phosphorothioate thymidine 149 TTTTACCCUG AUGGUAGACG CCGGGGUGTT TTT 33 33 base pairs nucleic acid single linear All A′s, C′s, G′s, T′s are 2′ deoxy-nucleotide derivatives modified base 1..4, 29..33 T at positions 1-4 and 29-33 are phosphorothioate thymidine 150 TTTTACCCTG ATGGTAGACG TTGGGGTGTT TTT 33 33 base pairs nucleic acid single linear All A′s, C′s, G′s, U′s are 2′OMe- nucleotide derivatives modified base 1..4, 29..33 T′s at positions 1-4 and 29-33 are 2′ deoxy phosphorothioate thymidine 151 TTTTACCCUG AUGGUAGACG CCGGGGUGTT TTT 33 33 base pairs nucleic acid single linear modified base 6..8, 19, 21..22 C at positions 6-8, 19 and 21-22 are 2′NH2 cytosine modified base 9, 12, 15, 27 U at positions 9, 12, 15 and 27 are 2′NH2 uracil modified base 5, 16 A at positions 5 and 16 are 2′OMe adenine modified base 13, 17, 20, 23..26, 28 G at positions 13, 17, 20, 23-26 and 28 are 2′OMe guanosine modified base 1..4, 29..33 T′s at positions 1-4 and 29-33 are 2′ deoxy phosphorothioate thymidine 152 TTTTACCCUG AUGGUAGACG CCGGGGUGTT TTT 33 33 base pairs nucleic acid single linear modified base 6..8, 19, 21..22 C at positions 6-8, 19 and 21-22 are 2′NH2 cytosine modified base 5, 8, 11, 23 U at positions 9, 12, 15 and 27 are 2′NH2 uracil modified base 5, 16 A at positions 5 and 16 are 2′OMe adenine modified base 13, 17, 20, 24..26, 28 G at positions 13, 17, 20, 24-26 and 28 are 2′OMe guanosine modified base 1..4, 29..33 T′s at positions 1-4 and 29-33 are 2′ deoxy phosphorothioate thymidine 153 TTTTACCCUG AUGGUAGACG CCGGGGUGTT TTT 33 33 base pairs nucleic acid single linear modified base 6..8, 19, 21..22 C at positions 6-8, 19 and 21-22 are 2′NH2 cytosine modified base 9, 12, 15, 27 U at positions 9, 12, 15 and 27 are 2′NH2 uracil modified base 5, 16 A at positions 5 and 16 are 2′OMe adenine modified base 13, 17, 20, 23..26, 28 G at positions 13, 17, 20, 23-26 and 28 are 2′OMe guanosine modified base 11 A at position 11 is phosphorothioate adenine modified base 1..4, 29..33 T′s at positions 1-4 and 29-33 are 2′ deoxy phosphorothioate thymidine 154 TTTTACCCUG AUGGUAGACG CCGGGGUGTT TTT 33 24 base pairs nucleic acid single linear modified base 2..4, 15, 17..18 C at positions 2-4, 15 and 17-18 are 2′NH2 cytosine modified base 5, 8, 11, 23 U at positions 5, 8, 11 and 23 are 2′NH2 uracil modified base G at position 9 is a 21 misture of 2′OMe guanosine and 2′OH guanosine modified base 12, 14 A at positions 12 and 14 are a 21 mixture of 2′OMe adenine and 2′OH adenine 155 ACCCUGAUGG UAGACGCCGG GGUG 24 24 base pairs nucleic acid single linear modified base 2..4, 15, 17..18 C at positions 2-4, 15 and 17-18 are 2′NH2 cytosine modified base 5, 8, 11, 23 U at positions 5, 8, 11 and 23 are 2′NH2 uracil modified base 1, 7 A at positions 1 and 7 are a 21 mixture of 2′OMe adenine and 2′OH adenine modified base 19, 21 G at positions 19 and 21 are a 21 mixture of 2′OMe guanosine and 2′OH guanosine 156 ACCCUGAUGG UAGACGCCGG GGUG 24 24 base pairs nucleic acid single linear modified base 2..4, 15, 17..18 C at positions 2-4, 15 and 17-18 are 2′NH2 cytosine modified base 5, 8, 11, 23 U at positions 5, 8, 11 and 23 are 2′NH2 uracil modified base 6, 20, 24 G at positions 6, 20, and 24 are a 21 mixture of 2′OMe guanosine and 2′OH guanosine 157 ACCCUGAUGG UAGACGCCGG GGUG 24 24 base pairs nucleic acid single linear modified base 2..4, 15, 17..18 C at positions 2-4, 15 and 17-18 are 2′NH2 cytosine modified base 5, 8, 11, 23 U at positions 5, 8, 11 and 23 are 2′NH2 uracil modified base 10, 13, 16 G at positions 10, 13 and 16 are a 21 mixture of 2′OMe guanosine and 2′OH guanosine 158 ACCCUGAUGG UAGACGCCGG GGUG 24 86 base pairs nucleic acid single linear 159 ATCCGCCTGA TTAGCGATAC TACAACGGCG TGGAAGACTA GAGTGCAGCC 50 GAACGCATCT AACTTGAGCA AAATCACCTG CAGGGG 86 86 base pairs nucleic acid single linear 160 ATCCGCCTGA TTAGCGATAC TACGCTACAA GTCCGCTGTG GTAGACAAGA 50 GTGCAGGCAA GACTTGAGCA AAATCACCTG CAGGGG 86 85 base pairs nucleic acid single linear 161 ATCCGCCTGA TTAGCGATAC TAGGCCCGTC GAAGNTAGAG CGCAGGGCCC 50 CAAAATACCG ACTTGAGCAA AATCACCTGC AGGGG 85 86 base pairs nucleic acid single linear 162 ATCCGCCTGA TTAGCGATAC TGTACCATCC ACGGTTTACG TGGACAAGAG 50 GGCCCTGGTA CACTTGAGCA AAATCACCTG CAGGGG 86 86 base pairs nucleic acid single linear 163 ATCCGCCTGA TTAGCGATAC TTCACTACAA GTCCGCCGTG GTAGACAAGA 50 GTGCAGGCAA GACTTGAGCA AAATCACCTG CAGGGG 86 85 base pairs nucleic acid single linear 164 ATCCGCCTGA TTAGCGATAC TACCGCTGTG TAGTTCCTTT AGGACTAGAG 50 GGCCGCCTAC ACTTGAGCAA AATCACCTGC AGGGG 85 85 base pairs nucleic acid single linear 165 ATCCGCCTGA TTAGCGATAC TTAGGCTTGA CGTCTTCTAG ACTAGAGTGC 50 AGTCAAACCC ACTTGAGCAA AATCACCTGC AGGGG 85 86 base pairs nucleic acid single linear 166 ATCCGCCTGA TTAGCGATAC TTGCAGGTCG ACTCTAGAGG ATCCCCGGGT 50 ACCGAGCTCG AACTTGAGCA AAATCACCTG CAGGGG 86 77 base pairs nucleic acid single linear 167 ATCCGCCTGA TTAGCGATAC TACGGTTTAC GTGGACAAGA GGGCCCTGGT 50 ACACTTGAGC AAAATCACCT GCAGGGG 77 86 base pairs nucleic acid single linear 168 ATCCGCCTGA TTAGCGATAC TGGTGGACTA GAGGNCAGCA AACGATCCTT 50 GGTTCGCGTC CACTTGAGCA AAATCACCTG CAGGGG 86 86 base pairs nucleic acid single linear 169 ATCCGCCTGA TTAGCGATAC TTCAAGCACT CCCGTCTTCC AGACAAGAGT 50 GCAGGGCCTC TACTTGAGCA AAATCACCTG CAGGGG 86 85 base pairs nucleic acid single linear 170 ATCCGCCTGA TTAGCGATAC TCGTGATGGA CAAGAGGGCC CTATCCACCG 50 GATATCCGTC ACTTGAGCAA AATCACCTGC AGGGG 85 85 base pairs nucleic acid single linear 171 ATCCGCCTGA TTAGCGATAC TCAAGCAGTG CCCGTCTTCC AGACAAGAGT 50 GCAGGCCTCT ACTTGAGCAA AATCACCTGC AGGGG 85 86 base pairs nucleic acid single linear 172 ATCCGCCTGA TTAGCGATAC TTGATCCACC GTTTATAGTC CGTGGTAGAC 50 AAGAGTGCAG GACTTGAGCA AAATCACCTG CAGGGG 86 85 base pairs nucleic acid single linear 173 ATCCGCCTGA TTAGCGATAC TAACACACAA GAGGACAGTT ACAGGTAACA 50 TCCGCTCAGG ACTTGAGCAA AATCACCTGC AGGGG 85 83 base pairs nucleic acid single linear 174 ATCCGCCTGA TTAGCGATAC TAGTGGCGTC TATAGACAAG AGTGCAGCCC 50 GAGTTTCAAC TTGAGCAAAA TCACCTGCAG GGG 83 84 base pairs nucleic acid single linear 175 ATCCGCCTGA TTAGCGATAC TCCACAAGAG GGCAGCAAGT GTACAACTAC 50 AGCGTCCGGA CTTGAGCAAA ATCACCTGCA GGGG 84 79 base pairs nucleic acid single linear 176 CTACCTACGA TCTGACTAGC GCAGGGCCAC GTCTATTTAG ACTAGAGTGC 50 AGTGGTTCGC TTACTCTCAT GTAGTTCCT 79 86 base pairs nucleic acid single linear 177 CTACCTACGA TCTGACTAGC ACGGTCCAAA GGTTTCCCAT CCGTGGACTA 50 GAGGGCACGT GCTTAGCTTA CTCTCATGTA GTTCCT 86 86 base pairs nucleic acid single linear 178 CTACCTACGA TCTGACTAGC CCGTCGCGTG ACTATAACCA CACGCAGACT 50 AGAGTGCAGG GCTTAGCTTA CTCTCATGTA GTTCCT 86 80 base pairs nucleic acid single linear 179 CTACCTACGA TCTGACTAGC CCGAATGGGG CTGCGACTGC AGTGGACGTC 50 ACGTCGTTAG CTTACTCTCA TGTAGTTCCT 80 80 base pairs nucleic acid single linear 180 CTACCTACGA TCTGACTAGC ACGCAAGAGA GTCNCCGAAT GCAGTCTCAG 50 CCGCTAACAG CTTACTCTCA TGTAGTTCCT 80 84 base pairs nucleic acid single linear 181 ATCCGCCTGA TTAGCGATAC TCANNNCACT GCAAGCAATT GTGGCCCAAA 50 GGGCTGAGTA CTTGAGCAAA ATCACCTGCA GGGG 84 86 base pairs nucleic acid single linear 182 ATCCGCCTGA TTAGCGATAC TGCTCGCTTA CAAAAGGGAG CCACTGTAGC 50 CCAGACTGGA CACTTGAGCA AAATCACCTG CAGGGG 86 86 base pairs nucleic acid single linear 183 ATCCGCCTGA TTAGCGATAC TGGTTATGGT GTGGTTCCGA ATGGTGGGCA 50 AAGTAACGCT TACTTGAGCA AAATCACCTG CAGGGG 86 85 base pairs nucleic acid single linear 184 ATCCGCCTGA TTAGCGATAC TGCTTGTNGC TCCGAAGGGG CGCGTATCCA 50 AGGACGGTTC ACTTGAGCAA AATCACCTGC AGGGG 85 83 base pairs nucleic acid single linear 185 ATCCGCCTGA TTAGCGATAC TTATGGAGTG GTTCCGAATG GTGGGCAAAG 50 TAACGCTTAC TTGAGCAAAA TCACCTGCAG GGG 83 86 base pairs nucleic acid single linear 186 CTACCTACGA TCTGACTAGC TGCNNGCGGG CGGTTCTCCG GATGGGACCA 50 TAAGGCTTTA GCTTAGCTTA CTCTCATGTA GTTCCT 86 80 base pairs nucleic acid single linear 187 CTACCTACGA TCTGACTAGC ACAAGGGGTC CTGNNGAATG GGGGAATACG 50 CTAGCCGAAG CTTACTCTCA TGTAGTTCCT 80 86 base pairs nucleic acid single linear 188 CTACCTACGA TCTGACTAGC AACACGAGCA TGTGGGGTCC CTTCCGAATG 50 GGGGGTACAG GCTTAGCTTA CTCTCATGTA GTTCCT 86 86 base pairs nucleic acid single linear 189 CTACCTACGA TCTGACTAGC GAGGCATTAG GTCCGAATGG TAGTAATGCT 50 GTCGTGCCTT GCTTAGCTTA CTCTCATGTA GTTCCT 86 80 base pairs nucleic acid single Linear 190 CTACCTACGA TCTGACTAGC CCGAATGGGG CTGCGACTGC AGTGGACGTC 50 ACGTCGTTAG CTTACTCTCA TGTAGTTCCT 80 86 base pairs nucleic acid single linear 191 CTACCTACGA TCTGACTAGC GAGGAGGTGC GTTGTCCGAA GGGGTCGTTA 50 GTCACCTCGT GCTTAGCTTA CTCTCATGTA GTTCCT 86 81 base pairs nucleic acid single linear 192 CTACCTACGA TCTGACTAGC GCAAGGGGTC CTGCCGAATG GGGGAATACG 50 CTAGCCGAAA GCTTACTCTC ATGTAGTTCC T 81 71 base pairs nucleic acid single linear 193 CTACCTACGA TCTGACTAGC ATCCTTCCGA ATGGGGGAAA TGGCGNCCCA 50 GCTTACTCTC ATGTAGTTCC T 71 82 base pairs nucleic acid single linear 194 CTACCTACGA TCTGACTAGC ACGCAAGAGA GGTCNCCGAA TGGCAGTCTC 50 AGCCGCTAAC AGCTTACTCT CATGTAGTTC CT 82 86 base pairs nucleic acid single linear 195 CTACCTACGA TCTGACTAGC CACGATAATC CTCCGAAAGC GTTGTCCGAA 50 TGGGTCGTTA GCTTAGCTTA CTCTCATGTA GTTCCT 86 85 base pairs nucleic acid single linear 196 ATCCGCCTGA TTAGCGATAC TTATCACCCC CACTGGATAG AGCCGCAGCG 50 TGCCCCTACT ACTTGAGCAA AATCACCTGC AGGGG 85 86 base pairs nucleic acid single linear 197 ATCCGCCTGA TTAGCGATAC TGCCCACTGC ATAGAGGGAC GGTTGTTTCC 50 GCCCGGTGTT TACTTGAGCA AAATCACCTG CAGGGG 86 81 base pairs nucleic acid single linear 198 CTACCTACGA TCTGACTAGC GTGAAGGAGC CCCAACTGGA TAGAAGCCTT 50 AAGGCGGTGT GCTTACTCTC ATGTAGTTCC T 81 86 base pairs nucleic acid single linear 199 CTACCTACGA TCTGACTAGC CCACCGCAGA GTGTTACACC CCATAGGAGA 50 AGTCCGGATG GCTTAGCTTA CTCTCATGTA GTTCCT 86 87 base pairs nucleic acid single linear 200 CTACCTACGA TCTGACTAGC CCACTGCATA GAGAGTCGCA AGACACGGTG 50 CTTTATTCNC CGCTTAGCTT ACTCTCATGT AGTTCCT 87 85 base pairs nucleic acid single linear 201 CTACCTACGA TCTGACTAGC TGCCCCACTG GATAGAGTAG GAGGCCTAGC 50 CGACACGGTG CTTAGCTTAC TCTCATGTAG TTCCT 85 86 base pairs nucleic acid single linear 202 CTACCTACGA TCTGACTAGC CGAGGTCCCC CACTGGATAG AGTTGTTGAA 50 ACAACGGTGC GCTTAGCTTA CTCTCATGTA GTTCCT 86 86 base pairs nucleic acid single linear 203 CTACCTACGA TCTGACTAGC AACACTTCCC CACTGGATAG AGGCCTTTCG 50 CAGAGCCGGT GCTTAGCTTA CTCTCATGTA GTTCCT 86 86 base pairs nucleic acid single linear 204 CTACCTACGA TCTGACTAGC CCACTGCATA GAGAACTGGA TCGACGGTCC 50 AAAGTTCGGT GCTTAGCTTA CTCTCATGTA GTTCCT 86 89 base pairs nucleic acid single linear 205 CTACCTACGA TCTGACTAGC CCACTGCATA GAGATACTGG ATTCGACNNN 50 CCAAAGTTTC GGTGCTTAGC TTACTCTCAT GTAGTTCCT 89 86 base pairs nucleic acid single linear 206 CTACCTACGA TCTGACTAGC CCACTGCAGA GAGTCAACCT TACGANGCCA 50 AGGTTGCGGT GCTTAGCTTA CTCTCATGTA GTTCCT 86 85 base pairs nucleic acid single linear 207 ATCCGCCTGA TTAGCGATAC TTCTGCGAGA GACCTACTGG AACGTTTTGT 50 GATATTCACA ACTTGAGCAA AATCACCTGC AGGGG 85 85 base pairs nucleic acid single linear 208 ATCCGCCTGA TTAGCGATAC TATACACCCG GCGGGCCTAC CGGATCGTTG 50 ATTTCTCTCC ACTTGAGCAA AATCACCTGC AGGGG 85 86 base pairs nucleic acid single linear 209 ATCCGCCTGA TTAGCGATAC TACGCCCCCT GAGACCTACC GGAATNTTNT 50 CGCTAGGCCT AACTTGAGCA AAATCACCTG CAGGGG 86 86 base pairs nucleic acid single linear 210 ATCCGCCTGA TTAGCGATAC TGGGCATCTA ACCCAGACCT ACCGGAACGT 50 TATCGCTTGT GACTTGAGCA AAATCACCTG CAGGGG 86 83 base pairs nucleic acid single linear 211 ATCCGCCTGA TTAGCGATAC TGGTGTGAAC CAGACCTACN GGAACGTTAT 50 CGCTTGTGAC TTGAGCAAAA TCACCTGCAG GGG 83 86 base pairs nucleic acid single linear 212 ATCCGCCTGA TTAGCGATAC TCATCAGTAT TATATAACGG GAACCAACGG 50 CAAATGCTGA CACTTGAGCA AAATCACCTG CAGGGG 86 85 base pairs nucleic acid single linear 213 ATCCGCCTGA TTAGCGATAC TTCCNNGGGA GAATAGGGTT AGTCGGAGAA 50 GTTAATCGCT ACTTGAGCAA AATCACCTGC AGGGG 85 86 base pairs nucleic acid single linear 214 ATCCGCCTGA TTAGCGATAC TCGGGAACGT GTGGTTACNC GGCCTACTGG 50 ATTGTTTCCT GACTTGAGCA AAATCACCTG CAGGGG 86 81 base pairs nucleic acid single linear 215 ATCCGCCTGA TTAGCGATAC TGGTAGGTCC GGTGTGAAAG AGGTTCGCAT 50 CAGGTAACTT GAGCAAAATC ACCTGCAGGG G 81 85 base pairs nucleic acid single linear 216 ATCCGCCTGA TTAGCGATAC TCCTCAGGCA ACATAGTTGA GCATCGTATC 50 GATCCTGGAG ACTTGAGCAA AATCACCTGC AGGGG 85 84 base pairs nucleic acid single linear 217 ATCCGCCTGA TTAGCGATAC TTTGGCTTGA GTCCCGGGAC GCACTGTTGA 50 CAGTGGAGTA CTTGAGCAAA ATCACCTGCA GGGG 84 86 base pairs nucleic acid single linear 218 ATCCGCCTGA TTAGCGATAC TCAGCAGGTT AGTATAACGG GAACCAACGG 50 CAAATGCTGA CACTTGAGCA AAATCACCTG CAGGGG 86 86 base pairs nucleic acid single linear 219 CTACCTACGA TCTGACTAGC GCAAGGGCAT CTCGGAATCG GTTAATCTGA 50 CTTGCAATAC GCTTAGCTTA CTCTCATGTA GTTCCT 86 75 base pairs nucleic acid single linear 220 CTACCTACGA TCTGACTAGC GATCCACGAA GAAGCTTACT CTCATGTAGT 50 TCCAGCTTAC TCTCATGTAG TTCCT 75 40 base pairs nucleic acid single linear 221 GTACCATCCA CGGTTTACGT GGACAAGAGG GCCCTGGTAC 40 31 base pairs nucleic acid single linear 222 GTAGTTCCTT TAGGACTAGA GGGCCGCCTA C 31 38 base pairs nucleic acid single linear 223 TGGACTAGAG GNCAGCAAAC GATCCTTGGT TCGCGTCC 38 26 base pairs nucleic acid single linear 224 CCCGTCTTCC AGACAAGAGT GCAGGG 26 36 base pairs nucleic acid single linear 225 AGGGCCACGT CTATTTAGAC TAGAGTGCAG TGGTTC 36 36 base pairs nucleic acid single linear 226 GGAGGTGCGT TGTCCGAAGG GGTCGTTAGT CACCTC 36 40 base pairs nucleic acid single linear 227 GCAAGGGGTC CTGCCGAATG GGGGAATACG CTAGCCGAAA 40 45 base pairs nucleic acid single linear 228 CGAGGTCCCC CACTGGATAG AGTTGTTGAA ACAACGGTGC GCTTA 45 45 base pairs nucleic acid single linear 229 AACACTTCCC CACTGGATAG AGGCCTTTCG CAGAGCCGGT GCTTA 45 40 base pairs nucleic acid single linear 230 GGGCATCTAA CCCAGACCTA CCGGAACGTT ATCGCTTGTG 40 15 base pairs nucleic acid single linear 231 AGACAAGAGT GCAGG 15 15 base pairs nucleic acid single linear 232 GGACTAGAGG GCAGT 15 18 base pairs nucleic acid single linear 233 CTCCGAATGG GGGNAAAG 18 15 base pairs nucleic acid single linear 234 CCCCACTGGA TAGAG 15 15 base pairs nucleic acid single linear 235 CCCCACTGCA TAGAG 15 17 base pairs nucleic acid single linear 236 AGACCTACCG GAACGTT 17 86 base pairs nucleic acid single linear 237 ATCCGCCTGA TTAGCGATAC TNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 50 NNNNNNNNNN NACTTGAGCA AAATCACCTG CAGGGG 86 28 base pairs nucleic acid single linear modified base 1..3 N at positions 1-3 is biotin 238 NNNCCCCTGC AGGTGATTTT GCTCAAGT 28 21 base pairs nucleic acid single linear 239 ATCCGCCTGA TTAGCGATAC T 21 81 base pairs nucleic acid single linear 240 CTACCTACGA TCTGACTAGC NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 50 NNNNNNNNNN GCTTACTCTC ATGTAGTTCC T 81 25 base pairs nucleic acid single linear modified base 2, 4 N at positions 2 and 4 is biotin 241 ANANAGGAAC TACATGAGAG TAAGC 25 20 base pairs nucleic acid single linear 242 CTACCTACGA TCTGACTAGC 20 

What is claimed is:
 1. A method of inhibiting binding of vascular endothelial growth factor (VEGF) to its receptor, comprising: a) providing a nucleic acid ligand to VEGF; and b) contacting the nucleic acid ligand with said VEGF under conditions where a VEGF:nucleic acid binding pair is formed, whereby binding of VEGF to its receptor is inhibited.
 2. The method of claim 1, wherein the nucleic acid ligand to VEGF is identified according to a method comprising the steps of: a) preparing a candidate mixture of nucleic acids; b) contacting the candidate mixture with VEGF, wherein nucleic acids having an increased affinity to VEGF relative to the candidate mixture may be partitioned from the remainder of the candidate mixture; c) partitioning the increased affinity nucleic acids from the remainder of the candidate mixture; and d) amplifying the increased affinity nucleic acids to yield a ligand-enriched mixture of nucleic acids, whereby a VEGF nucleic acid ligand may be identified.
 3. The method of claim 1, wherein said VEGF nucleic acid ligand is a ribonucleic acid.
 4. The method of claim 1, wherein said VEGF nucleic acid ligand is a deoxyribonucleic acid.
 5. The method of claim 1, wherein said VEGF nucleic acid ligand comprises modified nucleotides.
 6. The method of claim 5, wherein said VEGF ligand is comprised of 2′-amino (2′-NH₂) modified nucleotides and 2′-O-methyl (2′-OMe) modified nucleotides.
 7. The method of claim 1, wherein said VEGF nucleic acid ligand is modified with a moiety that decreases the activity of endonucleases or exonucleases on the nucleic acid ligand relative to the unmodified nucleic acid ligand, without adversely affecting the binding affinity of said nucleic acid ligand.
 8. The method of claim 7, wherein said moiety comprises a phosphorothioate nucleotide.
 9. The method of claim 1, wherein said VEGF nucleic acid ligand is modified by a method comprising substituting nucleotide residues, wherein said residues may be chemically modified, and wherein the modification decreases the activity of endonucleases or exonucleases relative to the unmodified nucleic acid ligand, without adversely affecting the binding affinity of said nucleic acid ligand. 